EP3558026B1 - Verfahren zur verwendung von thermostabilen serinproteasen - Google Patents

Verfahren zur verwendung von thermostabilen serinproteasen

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Publication number
EP3558026B1
EP3558026B1 EP17832424.0A EP17832424A EP3558026B1 EP 3558026 B1 EP3558026 B1 EP 3558026B1 EP 17832424 A EP17832424 A EP 17832424A EP 3558026 B1 EP3558026 B1 EP 3558026B1
Authority
EP
European Patent Office
Prior art keywords
seed
protease
fermentation
sequence
starch
Prior art date
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EP17832424.0A
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English (en)
French (fr)
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EP3558026A1 (de
Inventor
Jacob Flyvholm Cramer
Marc Anton Bernhard Kolkman
Zhen Ma
Martijn Scheffers
Stepan Shipovskov
Marco VAN BRUSSEL-ZWIJNEN
Shukun Yu
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International N&H Denmark ApS
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International N&H Denmark ApS
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Priority to EP25201503.7A priority Critical patent/EP4644560A2/de
Publication of EP3558026A1 publication Critical patent/EP3558026A1/de
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C7/00—Preparation of wort
    • C12C7/04—Preparation or treatment of the mash
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/347—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of proteins from microorganisms or unicellular algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03008—3-Phytase (3.1.3.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the field relates to methods of using thermostable serine proteases.
  • Proteases also called peptidases or proteinases are enzymes capable of cleaving peptide bonds. Proteases have evolved multiple times, and different classes of proteases can perform the same reaction by completely different catalytic mechanisms. Proteases can be found in animals, plants, fungi, bacteria, archaea and viruses.
  • Proteolysis can be achieved by enzymes currently classified into six broad groups: aspartyl proteases, cysteine proteases, serine proteases, threonine proteases, glutamic proteases, and metalloproteases.
  • Serine proteases are a subgroup of carbonyl hydrolases comprising a diverse class of enzymes having a wide range of specificities and biological functions. Notwithstanding this functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: 1) the subtilisins; and 2) chymotrypsin-related serine proteases (e.g. trypsin).
  • the invention provides a method for hydrolyzing starch-containing material comprising:
  • the invention further provides a method for producing fermentation products from starch-containing material comprising:
  • Steps (b) and (c) can be performed sequentially or can be performed simultaneously.
  • thermostable serine protease/metric ton (MT) starch-containing material wherein said thermostable serine protease has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3 is used.
  • thermostable serine protease/MT starch-containing material wherein said thermostable serine protease has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3.
  • a nitrogen source such as but not limiting to, urea
  • the invention further provides a method for reducing viscosity of a liquefied starch-containing material which comprises contacting a slurry of starch-containing material with a thermostable serine protease having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3.
  • a thermostable serine protease having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3.
  • Contacting of the thermostable serine protease described herein with the slurry can be in combination with other enzymes, or to a slurry comprising enzymes other than the thermostable serine protease described herein.
  • the invention further provides a method for extracting oil from an oilseed crop comprising:
  • oilseed fraction can be from soybean, corn seed, rape seed, palm kernel, sunflower seed, safflower seed, coconut, peanut, cotton seed, sesame seed, flax seed, poppy seed, almond, hazelnut, walnut, evening primrose seed, grape seed, hemp seed, black currant seed, red raspberry seed, carrot seed, cumin seed, blueberry seed, cranberry seed, parsley seed, onion seed, pumpkin seed, apricot kernel, mustard seed, linseed, castor seed or jatropha.
  • oilseed fraction can comprise a protein fraction that is useful as a food, food ingredient, a food additive or food supplement.
  • a feed, feedstuff, feed additive composition, premix, food or grain product comprising oil obtained by the method described herein.
  • the invention further provides a method for hydrolyzing at least one food or animal by-product comprising:
  • the food by-product may be keratin-rich material selected from the group consisting of feather, hair and wool.
  • the invention further provides a method for hydrolyzing proteins in lignocellulosic biomass comprising
  • SEQ ID NO:1 sets forth the nucleotide sequence of Tce01961n, encoding the protease ME-3.
  • SEQ ID NO:2 sets forth amino acid sequence of the ME-3 preproenzyme encoded by Tce01961n.
  • SEQ ID NO:3 sets forth the amino acid sequence of the fully processed mature enzyme, ME-3 (186 amino acids).
  • SEQ ID NO:4 sets forth the sequence of the modified aprE signal peptide sequence fused to the codon-optimized DNA sequence encoding the pro-mature sequence of ME-3 protease.
  • SEQ ID NO:5 sets forth the terminator sequence added after the tetracycline resistance gene using the Bst EII and Eco RI sites to make the pHYT vector derived from pHY300PLK (Takara Bio Inc.).
  • SEQ ID NO:6 sets forth the linker sequence cloned into the Bam HI and Hin dIII sites of pHY300PLK.
  • SEQ ID NO:7 sets forth the amino acid sequence of Thermobifida fusca Tfpa preproenzyme.
  • SEQ ID NO:8 sets forth the amino acid sequence of the fully processed mature enzyme, Tfpa (186 amino acids).
  • SEQ ID NO:9 sets forth the amino acid sequence of Thermobifida halotolerans WP_068687914 peptidase S1, Thpa.
  • SEQ ID NO:10 sets forth the amino acid sequence of the predicted fully processed mature enzyme, Thermobifida halotolerans WP_068687914 peptidase S1, or Thpa (186 amino acids).
  • SEQ ID NO: 11 sets forth the amino acid sequence of Nocardiopsis potens WP_017594871.
  • SEQ ID NO: 12 sets forth the amino acid sequence of the predicted fully processed mature enzyme, Nocardiopsis potens WP_017594871 serine protease (185 amino acids).
  • the term “about” refers to a range of +/- 0.5 of the numerical value, unless the term is otherwise specifically defined in context.
  • the phrase a "pH value of about 6" refers to pH values of from 5.5 to 6.5, unless the pH value is specifically defined otherwise.
  • protea means a protein or polypeptide domain of derived from a microorganism, e.g., a fungus, bacterium, or from a plant or animal, and that has the ability to catalyze cleavage of peptide bonds at one or more of various positions of a protein backbone (e.g., E.C. 3.4).
  • the terms “protease”, “peptidase” and “proteinase” can be used interchangeably.
  • Proteases can be found in animals, plants, fungi, bacteria, archaea and viruses. Proteolysis can be achieved by enzymes currently classified into six broad groups: aspartyl proteases, cysteine proteases, serine proteases, threonine proteases, glutamic proteases, and metalloproteases.
  • thermoostable serine protease means a serine protease that is heat-stable.
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants. This term refers to any plant-based material including, but not limited to, grains, grasses, tubers, and roots and, more specifically, to wheat, barley, corn, rye, rice, sorghum, brans, cassaya, millet, potato, sweet potato, and tapioca.
  • liquefy refers to the process or product of converting starch to soluble dextrinized substrates (e.g., smaller polysaccharides).
  • milled is used herein to refer to plant material that has been reduced in size, such as by grinding, crushing, fractionating or any other means of particle size reduction. Milling includes dry or wet milling. “Dry milling” refers to the milling of whole dry grain. “Wet milling” refers to a process whereby grain is first soaked (steeped) in water to soften the grain.
  • hydrolysis refers to a chemical reaction or process in which a chemical compound is broken down by reaction with water.
  • Starch digesting enzymes hydrolyze starch into smaller units, i.e, smaller polysaccharides.
  • lignocellulosic refers to a composition comprising both lignin and cellulose. It may also contain hemicellulose.
  • lignocellulosic biomass refers to any lignocellulosic material and includes materials comprising cellulose, hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass can also comprise additional components, such as protein and/or lipid. Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Lignocellulosic biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste.
  • biomass examples include, but are not limited to, corn cobs, crop residues such as corn husks, corn stover, grasses (including Miscanthus), wheat straw, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum material, soybean plant material, components obtained from milling of grains or from using grains in production processes (such as DDGS: dried distillers grains with solubles), trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, empty palm fruit bunch, and energy cane.
  • energy cane refers to sugar cane that is bred for use in energy production. It is selected for a higher percentage of fiber than sugar.
  • pretreated lignocellulosic biomass refers to biomass which has been subjected to a physical, thermal and/or chemical treatment prior to saccharification.
  • ammonia pretreated lignocellulosic biomass refers to biomass which has been subjected at least to a pretreatment process employing ammonia.
  • ammonia pretreatment is a low ammonia pretreatment where biomass is contacted with an aqueous solution comprising ammonia to form a biomass-aqueous ammonia mixture where the ammonia concentration is sufficient to maintain alkaline pH of the biomassaqueous ammonia mixture but is less than about 12 weight percent relative to dry weight of biomass, and where dry weight of biomass is at least about 15 weight percent solids relative to the weight of the biomass-aqueous ammonia mixture, as disclosed in the U.S. Patent No. 7,932,063 , which is herein incorporated by reference.
  • the term "lignocellulosic biomass hydrolysate" refers to the product resulting from saccharification of lignocellulosic biomass.
  • the biomass may also be pretreated or pre-processed prior to saccharification.
  • saccharification and “saccharifying” refer to the process of converting polysaccharides to dextrose monomers using enzymes. Saccharification can refer to the conversion of polysaccharides in a liquefact. Saccharification products are, for example, glucose and other small (low molecular weight) oligosaccharides such as disaccharides and trisaccharides.
  • SSF simultaneous saccharification and fermentation
  • enzyme cocktail refers to a mixture or combination of at least two different enzymes, which make it more efficient and effective for any catalytic reaction.
  • transfer or “fermenting” refer to the process of transforming sugars from reduced plant material to produce as fermentation product.
  • fertilization product means a product produced by a process including a fermentation step using a fermenting organism.
  • viscosity means the resistance of a fluid to flow, i.e., the internal friction of a fluid.
  • phospholipase refers to an enzyme that hydrolyzes phospholipids into fatty acids and other lipophilic substances.
  • emulsion refers to a mixture of two or more liquids that are normally immiscible (unmixable or unblendable). In an emulsion, one liquid (the dispersed phase) is dispersed in the other (continuous phase).
  • examples of emulsions include vinaigrettes, homogenized milk, mayonnaise, and the like.
  • ⁇ refers to grain or a quantity of grain to be ground; ground grain or meal produced from a grinding.
  • wort refers to the unfermented liquor run-off following extracting the grist during mashing.
  • mash refers to an aqueous slurry of any starch and/or sugar containing plant material such as grist, e. g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, mixed with water later to be separated into wort and spent grains.
  • grist e. g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, mixed with water later to be separated into wort and spent grains.
  • beer is meant to comprise any fermented wort, produced by fermentation/brewing of a starch-containing plant material, thus in particular also beer produced exclusively from malt or adjunct, or any combination of malt and adjunct.
  • Beer can be made from a variety of starch and/or sugar containing plant material, often cereal grains and/or malt by essentially the same process. Grain starches are believed to be glucose homopolymers in which the glucose residues are linked by either alpha-1, 4- or alpha-1,6-bonds, with the former predominating.
  • by-product refers to a secondary product derived from a manufacturing process or chemical reaction. It is not the primary product or service being produced.
  • an animal includes all non-ruminant (including humans) and ruminant animals.
  • the animal is a non-ruminant animal, such as a horse and a mono-gastric animal.
  • mono-gastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chicken, broiler chicks, layers; fish such as salmon, trout, tilapia, catfish and carps; and crustaceans such as shrimps and prawns.
  • the animal is a ruminant animal including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and nilgai.
  • pathogen means any causative agent of disease.
  • causative agents can include, but are not limited to, bacterial, viral, fungal causative agents and the like.
  • a “feed” and a “food,” respectively, means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by a non-human animal and a human being, respectively.
  • the term "food” is used in a broad sense and covers food and food products for humans as well as food for non-human animals (i.e. a feed).
  • feed is used with reference to products that are fed to animals in the rearing of livestock.
  • feed and “animal feed” are used interchangeably.
  • DFM direct-fed microbial
  • a DFM can comprise one or more of such naturally occurring microorganisms such as bacterial strains. Categories of DFMs include Bacillus, Lactic Acid Bacteria and Yeasts. Thus, the term DFM encompasses one or more of the following: direct fed bacteria, direct fed yeast, direct fed yeast and combinations thereof.
  • Lactic Acid Bacteria are unique, gram-positive rods that form spores. These spores are very stable and can withstand environmental conditions such as heat, moisture and a range of pH. These spores germinate into active vegetative cells when ingested by an animal and can be used in meal and pelleted diets. Lactic Acid Bacteria are gram-positive cocci that produce lactic acid which are antagonistic to pathogens. Since Lactic Acid Bacteria appear to be somewhat heat-sensitive, they are not used in pelleted diets. Types of Lactic Acid Bacteria include Bifidobacterium, Lactobacillus and Streptococcus.
  • prebiotic means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of beneficial bacteria.
  • probiotic culture defines live microorganisms (including bacteria or yeasts for example) which, when for example ingested or locally applied in sufficient numbers, beneficially affects the host organism, i.e. by conferring one or more demonstrable health benefits on the host organism.
  • Probiotics may improve the microbial balance in one or more mucosal surfaces.
  • the mucosal surface may be the intestine, the urinary tract, the respiratory tract or the skin.
  • probiotic as used herein also encompasses live microorganisms that can stimulate the beneficial branches of the immune system and at the same time decrease the inflammatory reactions in a mucosal surface, for example the gut.
  • CFU colony forming units
  • isolated means a substance in a form or environment that does not occur in nature.
  • isolated substances include (1 ) any non- naturally occurring substance, (2) any substance including, but not limited to, any host cell, enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated.
  • isolated nucleic acid molecule refers to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
  • purified as applied to nucleic acids or polypeptides generally denotes a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
  • a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified.”
  • a purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight on a molar basis).
  • a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique.
  • enriched refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
  • the term "functional assay” refers to an assay that provides an indication of a protein's activity.
  • the term refers to assay systems in which a protein is analyzed for its ability to function in its usual capacity.
  • a functional assay involves determining the effectiveness of the protease to hydrolyze a proteinaceous substrate.
  • peptides refer to a polymer of amino acids joined together by peptide bonds.
  • a “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues.
  • the single and 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure.
  • the single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Mutations can be named by the one letter code for the parent amino acid, followed by a position number and then the one letter code for the variant amino acid.
  • signal sequence and “signal peptide” refer to a sequence of amino acid residues that may participate in the secretion or direct transport of the mature or precursor form of a protein.
  • the signal sequence is typically located N-terminal to the precursor or mature protein sequence.
  • the signal sequence may be endogenous or exogenous.
  • a signal sequence is normally absent from the mature protein.
  • a signal sequence is typically cleaved from the protein by a signal peptidase after the protein is transported.
  • precursor form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein.
  • the precursor may also have a "signal" sequence operably linked to the amino terminus of the prosequence.
  • the precursor may also have additional polypeptides that are involved in post-translational activity (e.g., polypeptides cleaved therefrom to leave the mature form of a protein or peptide).
  • regulatory sequence or "control sequence” are used interchangeably herein and refer to a segment of a nucleotide sequence which is capable of increasing or decreasing expression of specific genes within an organism.
  • regulatory sequences include, but are not limited to, promoters, signal sequence, operators and the like. As noted above, regulatory sequences can be operably linked in sense or antisense orientation to the coding sequence/gene of interest.
  • Promoter refers to DNA sequences that define where transcription of a gene by RNA polymerase begins. Promoter sequences are typically located directly upstream or at the 5' end of the transcription initiation site. Promoters may be derived in their entirety from a native or naturally occurring sequence, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell type or at different stages of development, or in response to different environmental or physiological conditions ("inducible promoters").
  • the "3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include sequences encoding regulatory signals capable of affecting mRNA processing or gene expression, such as termination of transcription.
  • transformation refers to the transfer or introduction of a nucleic acid molecule into a host organism.
  • the nucleic acid molecule may be introduced as a linear or circular form of DNA.
  • the nucleic acid molecule may be a plasmid that replicates autonomously, or it may integrate into the genome of a production host. Production hosts containing the transformed nucleic acid are referred to as “transformed” or “recombinant” or “transgenic” organisms or “transformants”.
  • recombinant refers to an artificial combination of two otherwise separated segments of nucleic acid sequences, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. For example, DNA in which one or more segments or genes have been inserted, either naturally or by laboratory manipulation, from a different molecule, from another part of the same molecule, or an artificial sequence, resulting in the introduction of a new sequence in a gene and subsequently in an organism.
  • the terms “recombinant”, “transgenic”, “transformed”, “engineered” or “modified for exogenous gene expression” are used interchangeably herein.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not all found together in nature.
  • a construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
  • a plasmid vector can be used.
  • the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells.
  • the skilled artisan will also recognize that different independent transformation events may result in different levels and patterns of expression ( Jones et al., (1985) EMBO J 4:2411-2418 ; De Almeida et al., (1989) Mol Gen Genetics 218:78-86 ), and thus that multiple events are typically screened in order to obtain lines displaying the desired expression level and pattern.
  • Such screening may be accomplished standard molecular biological, biochemical, and other assays including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysis of protein expression, enzyme or activity assays, and/or phenotypic analysis.
  • Southern analysis of DNA Northern analysis of mRNA expression, PCR, real time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysis of protein expression, enzyme or activity assays, and/or phenotypic analysis.
  • production host refers to any organism, or cell thereof, whether human or non-human into which a recombinant construct can be stably or transiently introduced in order to express a gene. This term encompasses any progeny of a parent cell, which is not identical to the parent cell due to mutations that occur during propagation.
  • identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the number of matching nucleotides or amino acids between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988 ); Biocomputing: Informatics and Genome Projects (Smith, D.
  • % identity or percent identity or “PID” refers to protein sequence identity. Percent identity may be determined using standard techniques known in the art. Useful algorithms include the BLAST algorithms ( See, Altschul et al., J Mol Biol, 215:403-410, 1990 ; and Karlin and Altschul, Proc Natl Acad Sci USA, 90:5873-5787, 1993 ). The BLAST program uses several search parameters, most of which are set to the default values.
  • NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity but is not recommended for query sequences of less than 20 residues ( Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997 ; and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001 ).
  • a percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "reference" sequence including any gaps created by the program for optimal/maximum alignment.
  • BLAST algorithms refer to the "reference" sequence as the "query" sequence.
  • homologous proteins or “homologous proteases” refers to proteins that have distinct similarity in primary, secondary, and/or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins are aligned. Homologous search of protein sequences can be done using BLASTP and PSI-BLAST from NCBI BLAST with threshold (E-value cut-off) at 0.001. ( Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI BLAST a new generation of protein database search programs. Nucleic Acids Res 1997 Set 1;25(17):3389-402 ). Using this information, proteins sequences can be grouped. A phylogenetic tree can be built using the amino acid sequences.
  • Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), the AlignX program of Vector NTI v. 7.0 (Informax, Inc., Bethesda, MD), or the EMBOSS Open Software Suite (EMBL-EBI; Rice et al., Trends in Genetics 16, (6):276-277 (2000 )).
  • Multiple alignment of the sequences can be performed using the CLUSTAL method (such as CLUSTALW; for example, version 1.83) of alignment ( Higgins and Sharp, CABIOS, 5:151-153 (1989 ); Higgins et al., Nucleic Acids Res.
  • a fast or slow alignment is used with the default settings where a slow alignment.
  • polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain aspects. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein may be used in certain embodiments.
  • a variant polypeptide sequence or polynucleotide sequence in certain embodiments can have at least 60%, 61%, 62%,63%,64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein.
  • the variant amino acid sequence or polynucleotide sequence has the same function of the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function of the disclosed sequence.
  • variant refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally-occurring or man-made substitutions, insertions, or deletions of an amino acid.
  • variant refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.
  • Plasmid refers to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of double-stranded DNA.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences, in linear or circular form, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a polynucleotide of interest into a cell.
  • Transformation cassette refers to a specific vector containing a gene and having elements in addition to the gene that facilitates transformation of a particular host cell.
  • expression cassette and “expression vector are used interchangeably herein and refer to a specific vector containing a gene and having elements in addition to the gene that allow for expression of that gene in a host.
  • expression refers to the production of a functional endproduct (e.g., an mRNA or a protein) in either precursor or mature form. Expression may also refer to translation of mRNA into a polypeptide.
  • a functional endproduct e.g., an mRNA or a protein
  • Expression of a gene involves transcription of the gene and translation of the mRNA into a precursor or mature protein.
  • “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
  • Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
  • Stable transformation refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance.
  • transient transformation refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance.
  • Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms
  • the expression vector can be one of any number of vectors or cassettes useful for the transformation of suitable production hosts known in the art.
  • the vector or cassette will include sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors generally include a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. Both control regions can be derived from homologous genes to genes of a transformed production host cell and/or genes native to the production host, although such control regions need not be so derived.
  • Possible initiation control regions or promoters that can be included in the expression vector are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable, including but not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia ); and lac, araB, tet, trp, lPL, lPR, T7, tac, and trc (useful for expression in Escherichia coli ) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus.
  • the promoter is a constitutive or inducible promoter.
  • a "constitutive promoter” is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” or “repressible” promoter is a promoter that is active under environmental or developmental regulation.
  • promoters are inducible or repressible due to changes in environmental factors including but not limited to, carbon, nitrogen or other nutrient availability, temperature, pH, osmolarity, the presence of heavy metal(s), the concentration of inhibitor(s), stress, or a combination of the foregoing, as is known in the art.
  • the inducible or repressible promoters are inducible or repressible by metabolic factors, such as the level of certain carbon sources, the level of certain energy sources, the level of certain catabolites, or a combination of the foregoing as is known in the art.
  • the promoter is one that is native to the host cell.
  • the promoter is a native T. reesei promoter such as the cbh1 promoter which is deposited in GenBank under Accession Number D86235.
  • Microbiol., 63 :753-756 glucose repressible PCK1 promoter (see e.g., Leuker et al., (1997), Gene, 192:235-240 ), maltose inducible, glucoserepressible MET3 promoter (see Liu et al., (2006), Eukary. Cell, 5:638-649 ), pKi promoter and cpc1 promoter.
  • Other examples of useful promoters include promoters from A. awamori and A. niger glucoamylase genes (see e.g., Nunberg et al., (1984) Mol. Cell Biol.
  • T. reesei xln1 may be useful (see e.g., EPA 137280Al).
  • DNA fragments which control transcriptional termination may also be derived from various genes native to a preferred production host cell.
  • the inclusion of a termination control region is optional.
  • the expression vector includes a termination control region derived from the preferred host cell.
  • thermostable serine protease comprising:
  • thermostable serine protease optionally can be recovered from the production host.
  • thermostable serine protease-containing culture supernatant can be obtained by any of the methods described herein.
  • a polynucleotide encoding a thermostable serine protease can be manipulated in a variety of ways to provide for expression of the polynucleotide in a Bacillus host cell. Manipulation of the polynucleotide sequence prior to its insertion into a nucleic acid construct or vector may be desirable or necessary depending on the nucleic acid construct or vector or the Bacillus host cell. The techniques for modifying nucleotide sequences utilizing cloning methods are well known in the art.
  • thermostable serine protease include all components, which are necessary or advantageous for the expression of a thermostable serine protease.
  • Each control sequence may be native or foreign to the nucleotide sequence encoding the thermostable serine protease.
  • Such regulatory sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator.
  • Regulatory sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation or the regulatory sequences with the coding region of the nucleotide sequence encoding a thermostable serine protease.
  • a nucleic acid construct comprising a polynucleotide encoding a thermostable serine protease may be operably linked to one or more control sequences capable of directing the expression of the coding sequence in a Bacillus host cell under conditions compatible with the control sequences.
  • Each control sequence may be native or foreign to the polynucleotide encoding a thermostable serine protease.
  • control sequences include, but are not limited to, a leader, a promoter, a signal sequence, and a transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a thermostable serine protease.
  • the control sequence may be an appropriate promoter region, a nucleotide sequence that is recognized by a Bacillus host cell for expression of the polynucleotide encoding a thermostable serine protease.
  • the promoter region contains transcription control sequences that mediate the expression of a thermostable serine protease.
  • the promoter region may be any nucleotide sequence that shows transcriptional activity in the Bacillus host cell of choice and may be obtained from genes directing synthesis of extracellular or intracellular polypeptides having biological activity either homologous or heterologous to the Bacillus host cell.
  • the promoter region may comprise a single promoter or a combination of promoters. Where the promoter region comprises a combination of promoters, the promoters are preferably in tandem.
  • a promoter of the promoter region can be any promoter that can initiate transcription of a polynucleotide encoding a polypeptide having biological activity in a Bacillus host cell of interest.
  • the promoter may be native, foreign, or a combination thereof, to the nucleotide sequence encoding a polypeptide having biological activity.
  • Such a promoter can be obtained from genes directing synthesis of extracellular or intracellular polypeptides having biological activity either homologous or heterologous to the Bacillus host cell.
  • the promoter region comprises a promoter obtained from a bacterial source.
  • the promoter region comprises a promoter obtained from a Gram positive or Gram negative bacterium.
  • Gram positive bacteria include, but are not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus.
  • Gram negative bacteria include, but are not limited to, E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.
  • the promoter region may comprise a promoter obtained from a Bacillus strain (e.g., Bacillus agaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis ; or from a Streptomyces strain ( e.g., Streptomyces lividans or Streptomyces murinus ) .
  • Bacillus strain e.g., Bacillus agaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
  • promoters for directing transcription of a polynucleotide encoding a polypeptide having biological activity in the methods of the present disclosure are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus lentus or Bacillus clausii alkaline protease gene (aprH), Bacillus licheniformis alkaline protease gene (subtilisin Carlsberg gene), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis
  • cryIIIA tenebfionis CryIIIA gene or portions thereof, prokaryotic beta-lactamase gene ( Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731 ), and Bacillus megaterium xylA gene ( Rygus and Hillen, 1992, J. Bacteriol. 174: 3049-3055 ; Kim et al., 1996, Gene 181: 71-76 ).
  • Other examples are the promoter of the spo1 bacterial phage promoter and the tac promoter ( DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80:21-25 ).
  • the promoter region may comprise a promoter that is a "consensus” promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region.
  • the consensus promoter may be obtained from any promoter that can function in a Bacillus host cell.
  • the construction of a "consensus” promoter may be accomplished by site-directed mutagenesis using methods well known in the art to create a promoter that conforms more perfectly to the established consensus sequences for the "-10" and "-35" regions of the vegetative "sigma A-type” promoters for Bacillus subtilis ( Voskuil et al., 1995, Molecular Microbiology 17: 271-279 ).
  • a control sequence may also be a suitable transcription terminator sequence, such as a sequence recognized by a Bacillus host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding a thermostable serine protease. Any terminator that is functional in the Bacillus host cell may be used.
  • the control sequence may also be a suitable leader sequence, a non-translated region of a mRNA that is important for translation by a Bacillus host cell.
  • the leader sequence is operably linked to the 5' terminus of the nucleotide sequence directing synthesis of the polypeptide having biological activity. Any leader sequence that is functional in a Bacillus host cell of choice may be used in the present invention.
  • the control sequence may also be a mRNA stabilizing sequence.
  • mRNA stabilizing sequence is defined herein as a sequence located downstream of a promoter region and upstream of a coding sequence of a polynucleotide encoding a thermostable serine protease to which the promoter region is operably linked, such that all mRNAs synthesized from the promoter region may be processed to generate mRNA transcripts with a stabilizer sequence at the 5' end of the transcripts.
  • the mRNA processing/stabilizing sequence is complementary to the 3' extremity of bacterial 16S ribosomal RNA.
  • the mRNA processing/stabilizing sequence generates essentially single-size transcripts with a stabilizing sequence at the 5' end of the transcripts.
  • the mRNA processing/stabilizing sequence is preferably one, which is complementary to the 3' extremity of a bacterial 16S ribosomal RNA. See, U.S. Patent No. 6,255,076 and U.S. Patent No. 5,955,310 .
  • the nucleic acid construct can then be introduced into a Bacillus host cell using methods known in the art or those methods described herein for introducing and expressing a thermostable serine protease.
  • a nucleic acid construct comprising a DNA of interest encoding a protein of interest can also be constructed similarly as described above.
  • control sequence may also comprise a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of a polypeptide that can direct the expressed polypeptide into the cell's secretory pathway.
  • the signal peptide coding region may be native to the polypeptide or may be obtained from foreign sources.
  • the 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide.
  • the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to that portion of the coding sequence that encodes the secreted polypeptide.
  • the foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region.
  • the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the polypeptide relative to the natural signal peptide coding region normally associated with the coding sequence.
  • the signal peptide coding region may be obtained from an amylase or a protease gene from a Bacillus species. However, any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of a Bacillus host cell of choice may be used in the present invention.
  • An effective signal peptide coding region for a Bacillus host cell is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis prsA gene.
  • a polynucleotide construct comprising a nucleic acid encoding a thermostable serine protease construct comprising a nucleic acid encoding a polypeptide of interest (POI) can be constructed such that it is expressed by a host cell.
  • POI polypeptide of interest
  • Nucleic acids encoding proteins of interest can be incorporated into a vector, wherein the vector can be transferred into a host cell using well-known transformation techniques, such as those disclosed herein.
  • the vector may be any vector that can be transformed into and replicated within a host cell.
  • a vector comprising a nucleic acid encoding a POI can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector.
  • the vector also may be transformed into a Bacillus expression host of the disclosure, so that the protein encoding nucleic acid (e.g., an ORF) can be expressed as a functional protein.
  • a representative vector which can be modified with routine skill to comprise and express a nucleic acid encoding a POI is vector p2JM103BBI.
  • a polynucleotide encoding a thermostable serine protease or a POI can be operably linked to a suitable promoter, which allows transcription in the host cell.
  • the promoter may be any nucleic acid sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Means of assessing promoter activity/strength are routine for the skilled artisan.
  • Suitable promoters for directing the transcription of a polynucleotide sequence encoding comS1 polypeptide or a POI of the disclosure include the promoter of the lac operon of E . coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis alpha-amylase gene ( amyL ) , the promoters of the Bacillus stearothermophilus maltogenic amylase gene ( amyM ) , the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes, and the like.
  • a promoter for directing the transcription of a polynucleotide sequence encoding a POI can be a wild-type aprE promoter, a mutant aprE promoter or a consensus aprE promoter set forth in PCT International Publication No. WO2001/51643 .
  • a promoter for directing the transcription of a polynucleotide sequence encoding a POI is a wild-type spoVG promoter, a mutant spoVG promoter, or a consensus spoVG promoter (Frisby and Zuber, 1991).
  • a promoter for directing the transcription of the polynucleotide sequence encoding a thermostable serine protease or a POI is a ribosomal promoter such as a ribosomal RNA promoter or a ribosomal protein promoter.
  • the ribosomal RNA promoter can be a rrn promoter derived from B. subtilis, more particularly, the rrn promoter can be a rrnB, rrnI or rrnE ribosomal promoter from B. subtilis.
  • the ribosomal RNA promoter is a P2 rrnI promoter from B. subtilis set forth in PCT International Publication No. WO2013/086219 .
  • a suitable vector may further comprise a nucleic acid sequence enabling the vector to replicate in the host cell.
  • enabling sequences include the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, pIJ702, and the like.
  • a suitable vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis ; or a gene that confers antibiotic resistance such as, e.g., ampicillin resistance, kanamycin resistance, chloramphenicol resistance, tetracycline resistance and the like.
  • a selectable marker e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis ; or a gene that confers antibiotic resistance such as, e.g., ampicillin resistance, kanamycin resistance, chloramphenicol resistance, tetracycline resistance and the like.
  • a suitable expression vector typically includes components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes.
  • Expression vectors typically also comprise control nucleotide sequences such as, for example, promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene, one or more activator genes sequences, or the like.
  • a suitable expression vector may further comprise a sequence coding for an amino acid sequence capable of targeting the protein of interest to a host cell organelle such as a peroxisome, or to a particular host cell compartment.
  • a targeting sequence may be, for example, the amino acid sequence "SKL".
  • the nucleic acid sequence of the protein of interest can be operably linked to the control sequences in a suitable manner such that the expression takes place.
  • Protocols such as described herein, used to ligate the DNA construct encoding a protein of interest, promoters, terminators and/or other elements, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art.
  • An isolated cell is advantageously used as a host cell in the recombinant production of a POI.
  • the cell may be transformed with the DNA construct encoding the POI, conveniently by integrating the construct (in one or more copies) into the host chromosome. Integration is generally deemed an advantage, as the DNA sequence thus introduced is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed applying conventional methods, for example, by homologous or heterologous recombination. For example, PCT International Publication No. WO2002/14490 describes methods of Bacillus transformation, transformants thereof and libraries thereof. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • genes from expression hosts where the gene deficiency can be cured by an expression vector.
  • Known methods may be used to obtain a bacterial host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein.
  • Techniques for transformation of bacteria and culturing the bacteria are standard and well known in the art. They can be used to transform the improved hosts of the present invention for the production of recombinant proteins of interest.
  • Introduction of a DNA construct or vector into a host cell includes techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated and DEAE-Dextrin mediated transfection), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, gene gun or biolistic transformation and protoplast fusion, and the like. Transformation and expression methods for bacteria are also disclosed in Brigidi et al. (1990). A general transformation and expression protocol for protease deleted Bacillus strains is described in Ferrari et al. (U.S. Patent No. 5, 264,366 ).
  • nucleic acids into filamentous fungi such as Aspergillus spp., e.g., A. oryzae or A. niger, H. grisea, H. insolens, and T. reesei. are well known in the art.
  • a suitable procedure for transformation of Aspergillus host cells is described, for example, in EP238023 .
  • a suitable procedure for transformation of Trichoderma host cells is described, for example, in Steiger et al 2011, Appl. Environ. Microbiol. 77:114-121 .
  • the choice of a production host can be any suitable microorganism such as bacteria, fungi and algae.
  • thermostable serine protease typically, the choice will depend upon the gene encoding the thermostable serine protease and its source.
  • Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, (e.g. , lipofection mediated and DEAE-Dextrin mediated transfection); incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion.
  • Basic texts disclosing the general methods that can be used include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989 ); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990 ); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994 )).
  • the methods of transformation of the present invention may result in the stable integration of all or part of the transformation vector into the genome of a host cell, such as a filamentous fungal host cell.
  • transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated.
  • Fusarium include Bajar, Podila and Kolattukudy, (1991) Proc. Natl. Acad. Sci. USA 88: 8202-8212
  • Streptomyces include Hopwood et al., 1985, Genetic Manipulation of Streptomyces: Laboratory Manual, The John Innes Foundation, Norwich, UK and Fernandez-Abalos et al., Microbiol 149:1623 - 1632 (2003 ) and for Bacillus include Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, (1990) FEMS Microbiol. Lett. 55: 135-138 ).
  • any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra ) . Also of use is the Agrobacterium-mediated transfection method described in U.S. Patent No. 6,255,115 . It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the gene.
  • the transfected or transformed cells are cultured under conditions favoring expression of genes under control of the promoter sequences.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell and obtaining expression of an alpha-glucosidase polypeptide.
  • Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
  • thermostable serine polypeptide secreted from the host cells can be used, with minimal post-production processing, as a whole broth preparation.
  • post-transcriptional and/or post-translational modifications may be made.
  • One non-limiting example of a post-transcriptional and/or post-translational modification is "clipping" or "truncation" of a polypeptide. For example, this may result in taking a thermostable serine protease from an inactive or substantially inactive state to an active state as in the case of a pro-peptide undergoing further post-translational processing to a mature peptide having the enzymatic activity.
  • this clipping may result in taking a mature thermostable serine protease polypeptide and further removing N or C-terminal amino acids to generate truncated forms of the thermostable serine protease that retain enzymatic activity.
  • post-transcriptional or post-translational modifications include, but are not limited to, myristoylation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation.
  • the skilled person will appreciate that the type of post-transcriptional or post-translational modifications that a protein may undergo may depend on the host organism in which the protein is expressed.
  • a lipase and/or a phytase can be added to any one step of the fermentation process.
  • the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of a thermostable serine protease.
  • Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the alpha-glucosidase to be expressed or isolated.
  • the term "spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g ., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.
  • Host cells may be cultured under suitable conditions that allow expression of a thermostable serine protease.
  • Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or sophorose.
  • fungal cells are grown under batch or continuous fermentation conditions.
  • a classical batch fermentation is a closed system, where the composition of the medium is set at the beginning of the fermentation, and the composition is not altered during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In other words, the entire fermentation process takes place without addition of any components to the fermentation system throughout.
  • a batch fermentation qualifies as a "batch” with respect to the addition of the carbon source. Moreover, attempts are often made to control factors such as pH and oxygen concentration throughout the fermentation process. Typically, the metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high growth log phase and finally to a stationary phase, where growth rate is diminished or halted. Left untreated, cells in the stationary phase would eventually die. In general, cells in log phase are responsible for the bulk of production of product.
  • a suitable variation on the standard batch system is the "fed-batch fermentation" system. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses.
  • Fed-batch systems are useful when it is known that catabolite repression would inhibit the metabolism of the cells, and/or where it is desirable to have limited amounts of substrates in the fermentation medium. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors, such as pH, dissolved oxygen and the partial pressure of waste gases, such as CO2. Batch and fed-batch fermentations are well known in the art.
  • Continuous fermentation is another known method of fermentation. It is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant density, where cells are maintained primarily in log phase growth. Continuous fermentation allows for the modulation of one or more factors that affect cell growth and/or product concentration. For example, a limiting nutrient, such as the carbon source or nitrogen source, can be maintained at a fixed rate and all other parameters are allowed to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.
  • thermostable serine protease polypeptide of the invention Separation and concentration techniques are known in the art and conventional methods can be used to prepare a concentrated solution or broth comprising a thermostable serine protease polypeptide of the invention.
  • thermostable serine protease solution After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain a thermostable serine protease solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra- filtration, extraction, or chromatography, or the like, are generally used.
  • the enzyme-containing solution can be concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Examples of methods of enrichment and purification include but are not limited to rotary vacuum filtration and/or ultrafiltration.
  • thermostable serine protease-containing solution or broth may be concentrated until such time the enzyme activity of the concentrated a thermostable serine protease polypeptide-containing solution or broth is at a desired level.
  • Concentration may be performed using, e.g., a precipitation agent, such as a metal halide precipitation agent.
  • a precipitation agent such as a metal halide precipitation agent.
  • Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides and blends of two or more of these metal halides.
  • Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide and blends of two or more of these metal halides.
  • the metal halide precipitation agent, sodium chloride can also be used as a preservative.
  • thermostable serine protease polypeptides can be enriched or partially purified as generally described above by removing cells via flocculation with polymers.
  • the enzyme can be enriched or purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not need to be enriched or purified, and whole broth culture can be lysed and used without further treatment. The enzyme can then be processed, for example, into granules.
  • Thermostable serine proteases may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
  • Standard purification methods include, but are not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, immunological and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), extraction microfiltration, two phase separation and crystallization.
  • the protein of interest may be purified using a standard anti-protein of interest antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, Protein Purification (1982). The degree of purification necessary will vary depending on the use of the protein of interest. In some instances, no purification will be necessary.
  • Assays for detecting and measuring the enzymatic activity of an enzyme are well known.
  • Various assays for detecting and measuring activity of proteases are also known to those of ordinary skill in the art.
  • assays are available for measuring protease activity that are based on the release of acid-soluble peptides from casein or hemoglobin, measured as absorbance at 280 nm or colorimetrically using the Folin method, and hydrolysis of the dye-labeled azocasein, measured as absorbance at 440-450 nm
  • exemplary assays involve the solubilization of chromogenic substrates (See e.g., Ward, "Proteinases,” in Fogarty (ed.)., Microbial Enzymes and Biotechnology, Applied Science, London, [1983], pp. 251-317 ).
  • a protease detection assay method using highly labeled fluorescein isothiocyanate (FITC) casein as the substrate, a modified version of the procedure described by Twining [ Twining, S.S., (1984) "Fluorescein Isothiocyanate-Labeled Casein Assay for Proteolytic Enzymes" Anal. Biochem. 143:30-34 ] may also be used.
  • FRET Fluorescence Resonance Energy Transfer
  • FRET Fluorescence Activated FRET
  • substrates in which the fluorophore is separated from the quencher by a short peptide sequence containing the enzyme cleavage site. Proteolysis of the peptide results in fluorescence as the fluorophore and quencher are separated.
  • suitable methods See e.g., Wells et al., Nucleic Acids Res. 11:7911-7925 [1983] ; Christianson et al., Anal. Biochem. 223:119 -129 [1994] ; and Hsia et al., Anal Biochem. 242:221-227 [1999] ).
  • the enzyme cocktail may comprise at least one enzyme selected from the group consisting of alpha-amylase, amyloglucosidase, phytase, pullulanase, beta-glucanase, cellulase and xylanase.
  • Any of these enzymes can be used in an amount ranging from 0.5 to 500 micrograms/g feed or feedstock.
  • Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1.) hydrolyze internal alpha-1,4-glucosidic linkages in starch, largely at random to produce smaller molecular weight dextrans. These polypeptides are used, inter alia, in starch processing and in alcohol production. Any alpha-amylases can be used, e.g., those described in U.S. Patent Nos. 8,927,250 and 7,354,752 .
  • Amyloglucosidase catalyzes the hydrolysis of terminally 1,4-linked alpha-D-glucose residues successively from the non-reducing ends of maltooligo- and polysaccharides with release of beta-D-glucose. Any amyloglucosidase can be used.
  • Phytase refers to a protein or polypeptide which is capable of catalyzing the hydrolysis of phytate to (1) myo-inositol and/or (2) mono-, di-, tri-, tetra-, and/or penta-phosphatess thereof and (3) inorganic phosphate.
  • enzymes having catalytic activity as defined in Enzyme Commission EC number 3.1.3.8 or EC number 3.1.3.26. Any phytase can be used such as described in U.S. Patent Nos. 8,144,046 , 8,673,609 , and 8,053,221 .
  • Pullulanase (EC 3.2.1.41) is a specific kind of glucanase, an amylolytic exoenzyme that degrades pullan (a polysaccharide polymer consisting of maltotriose units, also known as alpha-1,4-; alpha-1,6-glucan. Thus, it is an example of a debranching enzyme.
  • Pullulanase is also known as pullulan-6-glucanohydrolase.
  • Pullulanases are generally secreted by a Bacillus species. For example, Bacillus deramificans ( US Patent No. 5,817,498; 1998 ), Bacillus acidopullulyticus ( European Patent No.
  • Enzymes having pullulanase activity used commercially are produced, for example, from Bacillus species (trade name OPITMAX ® 1-100 from DuPont-Genencor and Promozyme ® D2 fro Novozymes).
  • Other examples of debranching enzymes include, but are not limited to, iso-amylase from Sulfolobus solfataricus, Pseudomonas sp. and thermostable pullulanase from Fervidobacterium nodosum (e.f., WO2010/76113 ).
  • the iso-amylase from Pseudomonas sp. is available as purified enzyme from Megazyme International. Any pullulanase can be used.
  • Glucanases are enzymes that break down a glucan, a polysaccharide made several glucose sub-units. As they perform hydrolysis of the glucosidic bond, they are hydrolases.
  • Beta-glucanase enzymes (EC 3.2.1.4) digests fiber. It helps in the breakdown of plant walls (cellulose).
  • Cellulases are any of several enzymes produced by fungi, bacteria and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides. The name is also used for any naturally-occurring mixture or complex of various such enzymes, that act serially or synergistically to decompose cellulosic material. Any cellulases can be used.
  • Xylanase (EC 3.2.1.8) is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into xylose, those breaking down hemicellulose, one of the major components of plant cell walls. Any xylanases can be used.
  • Starch or amylum is a polymeric carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. Starch can be hydrolyzed into simpler carbohydrates by acids, various enzymes or a combination of the two.
  • starch is converted into sugars, for example by malting, and fermented to produce ethanol in the manufacture of beer, whiskey and biofuel.
  • the starting material is generally selected based on the desired fermentation product.
  • starch-containing materials include but are not limited to, whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or mixtures thereof or starches derived therefrom, or cereals.
  • the starting material can be a dry solid, such as but not limiting to a dry solid of a feed or feedstock as described herein. Contemplated are also waxy and non-waxy types or corn and barley.
  • Starch-containing materials used for ethanol production is corn or wheat. Starch is initially collected from plant grains using either a wet milling, a dry milling or a dry grind process.
  • a substrate comprising plant material is reduced or milled by methods known in the art.
  • Plant material can be obtained from: wheat, corn, rye, sorghum (milo), rice, millet, barley, triticale, cassava (tapioca), potato, sweet potato, sugar beets, sugarcane, and legumes such as soybean and peas.
  • Preferred plant material includes corn, barley, wheat, rice, milo and combinations thereof.
  • Plant material can include hybrid varieties and genetically modified varieties (e.g. transgenic corn, barley or soybeans comprising heterologous genes).
  • any part of the plant containing starch can be used to produce the liquefact, including but not limited to, plant parts such as leaves, stems, hulls, husks, tubers, cobs, grains and the like.
  • Preferred whole grains include corn, wheat, rye, barley, sorghum and combinations thereof.
  • starch can be obtained from fractionated cereal grains including fiber, endosperm and/or germ components. Methods for fractionating plant material, such as corn and wheat, are known in the art.
  • plant material obtained from different sources can be mixed together (e.g. corn and milo or corn and barley).
  • the plant material whether reduced by milling or other means, will be combined with a solution resulting in a slurry comprising starch substrate.
  • the slurry can include a side stream from starch processing such as backset.
  • the slurry will comprise 15 - 55% ds (e.g., 20 - 50%, 25 - 45%, 25 - 40%, and 20 - 35%).
  • the slurry can comprise 10% to 60% of backset.
  • the slurry comprising the reduced plant material can be subject to a liquefaction process wherein an alpha amylase can be added during the liquefaction step. This results in a liquefact. To produce the liquefact, a single or split dose of an alpha amylase can be added to the slurry.
  • One skilled in the art can readily determine the effective dosage of alpha amylase to be used in the liquefaction processes.
  • the amount of alpha amylase used for liquefaction is an amount effective to cause liquefaction of a majority of the starch. In other embodiments, the amount is effective to enable liquefaction of greater than 40% of the starch, including 50%, 60%, 70%, 80%, 90%, and 100%. In some embodiments, the range will be 0.05 to 50 AAU/g ds (alpha-amylase units per gram of dry solids (e.g., feed or feedstock, such as corn), also 0.1 to 20 AAU/gDS and also 1.0 to 10 AAU/gDS. In further embodiments, the alpha amylase dosage will be in the range of 0.01 to 10.0 kg/metric ton (MT)ds; also 0.05 to 5.0 kg/MT ds; and also 0.1 to 4.0 kg/MT ds.
  • MT weight/metric ton
  • An alpha amylase can be added at a temperature of 0 to 30°C below the starch gelatinization temperature of the granular starch of the reduced plant material. This temperature can be 0 to 25°C, 0 to 20°C, 0 to 15°C and 0 to 10°C below the starch gelatinization temperature. This specific value will vary and depends on the type of grain comprising the slurry. For example, the starch gelatinization temperature of corn is generally higher than the starch gelatinization temperature of rye or wheat.
  • the slurry will be maintained at a pH range of about 3.0 to less than 6.5, also at a pH range of 4.0 to less than 6.2, also at a pH range of about 4.5 to less than 6.0 and preferably at a pH range of about 5.0 to 6.0 ( e.g . about 5.4 to 5.8), and the milled grain in the slurry will be contacted with the enzyme composition for a period of time of 2 minutes to 8 hours ( e.g., 5 mins to 3 hrs; 15 mins to 2.5 hrs and 30 min to 2 hrs).
  • the incubated substrate will be liquefied by exposing the incubated substrate to an increase in temperature such as 0 to 55°C above the starch gelatinization temperature. (e.g . to 65°C to 120°C, 70°C to 110°C, 70°C to 90°C) for a period of time of 2 minutes to 8 hours ( e.g. , 2 minutes to 6 hrs, 5 minutes to 4 hours and preferably 1hr to 2 hrs) at a pH of about 4.0 to 6.5.
  • the temperature can be raised to a temperature to between about 85-90°C and a single dose of alpha amylase can be used.
  • a second dose of alpha amylase can be added after the temperature returns to normal.
  • the temperature can be raised to between about 105 and 140°C and a split dose of alpha amylase can be used with one part being added before raising the temperature and the other part added after the temperature has been brought down to at least below 105°C, including below 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, and 91°C, but preferably below 90°C.
  • the resulting liquefact is cooled before saccharification.
  • a split dose means that the total dose in desired ratio is added in more than one portion, including two portions or three portions.
  • one portion of the total dose is added at the beginning and a second portion is added at a specified time in the process.
  • at least a portion of the dose is added at the beginning of the saccharification (or SSF) to begin the saccharification process.
  • each enzyme in the enzyme composition can be added to the liquefact separately, but simultaneously or close enough in time such that the activity ratio is maintained.
  • the enzyme blend composition comprising a glucoamylase, an acid stable alpha amylase, and an acid fungal protease can be added during one or both of the saccharification and fermentation.
  • the ratio of the glucoamylase, an acid stable alpha amylase, and an acid fungal protease is preferably about 1:1.5:0.1 to about 1:8:1, and more preferably about 1:2:0.2 to 1:5: 0.6, as measured by GAU:SSU:SAPU.
  • the saccharification or SSF process typically comprises the addition of urea or ammonia used as a nitrogen source for the host organism (such as but not limiting to Yeast).
  • the addition of the nitrogen source (such as urea) during saccharification or SSF typically ranges between 200-1000 ppm, such as at least 200, 300, 400, 500, 600, 700, 800, 900 up to 1000 ppm urea.
  • Another rich nitrogen source in an ethanol plant are the corn proteins present in the corn kernel. This nitrogen source is made available for the fermenting organism when it is hydrolysed into smaller fragments like small peptides and amino acids.
  • thermostable serine protease such as ME3
  • the need for adding a nitrogen source during the saccharification or SSF process is reduced by at least 30%, 40%, 50, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or completely eliminated (100% reduction).
  • thermostable serine protease that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 (such as but not limiting to ME-3 described herein) can hydrolyze corn proteins to such extend that it enables the fermenting organism to use corn proteins as a nitrogen source. Consequently, less urea or no urea at all can be added to the SSF. The strong reduction or elimination of a nitrogen source enables the plant to run at a lower cost.
  • thermostable serine protease that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 (such as but not limiting to ME-3 described herein) in the fermentation process may not be needed.
  • the saccharification process can last for 12 to 120 hours. However, it is common to perform a saccharification for 30 minutes to 2 hours and then complete the saccharification during fermentation. Sometimes this is referred to as simultaneous saccharification and fermentation (SSF). Saccharification is commonly carried out at temperatures of 30 to 65°C and typically at pH of 3.0 to 5.0, including 4.0 to 5.0. The saccharification can result in the production of fermentable sugars.
  • SSF simultaneous saccharification and fermentation
  • the fermentable sugars are subjected to fermentation with fermenting microorganisms.
  • the contacting step and the fermenting step can be performed simultaneously in the same reaction vessel or sequentially.
  • fermentation processes are described in The Alcohol Textbook 3rd ED, A Reference for the Beverage, Fuel and Industrial Alcohol Industries, Eds Jacques et al., (1999) Nottingham University Press, UK .
  • the method further comprises using the fermentable sugars (dextrin e.g. glucose) as a fermentation feedstock in microbial fermentations under suitable fermentation conditions to obtain end-products, such as alcohol (e.g., ethanol), organic acids (e.g., succinic acid, lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, DKG, KLG) amino acids (e.g., lysine), proteins (e.g., antibodies and fragment thereof).
  • alcohol e.g., ethanol
  • organic acids e.g., succinic acid, lactic acid
  • sugar alcohols e.g., glycerol
  • ascorbic acid intermediates e.g., gluconate, DKG, KLG
  • amino acids e.g., lysine
  • proteins e.g., antibodies and fragment thereof.
  • the fermentable sugars can be fermented with a yeast at temperatures in the range of 15 to 40°C, 20 to 38°C, and also 25 to 35°C; at a pH range of pH 3.0 to 6.5; also pH 3.0 to 6.0; pH 3.0 to 5.5, pH 3.5 to 5.0 and also pH 3.5 to 4.5 for a period of time of 5 hrs to 120 hours, preferably 12 to 120 and more preferably from 24 to 90 hours to produce an alcohol product, preferably ethanol.
  • Yeast cells are generally supplied in amounts of 10 4 to 10 12 , and preferably from 10 7 to 10 10 viable yeast count per ml of fermentation broth.
  • the fermentation will include in addition to a fermenting microorganism (e.g. yeast) nutrients, optionally acid and additional enzymes.
  • fermentation media will contain supplements including but not limited to vitamins (e.g. biotin, folic acid, nicotinic acid, riboflavin), cofactors, and macro and micro-nutrients and salts (e.g. (NH4) 2 SO 4 ; K 2 HPO 4 ; NaCl; MgSO 4 ; H 3 BO 3 ; ZnCl 2 ; and CaCl 2 ).
  • the milled plant material includes barley, milo, corn and combinations thereof, and the contacting and fermenting steps are conducted simultaneously at a pH range of 3.5 to 5.5, a temperature range of 30 - 45°C, and for a period of time of 48 to 90 hrs, wherein at least 50% of the starch is solubilized.
  • One end product of a fermentation process can be an alcohol product, e.g. ethanol.
  • Other end products can be the fermentation co-products such as distillers dried grains (DDG) and distiller's dried grain plus solubles (DDGS), which can be used as an animal feed.
  • DDG distillers dried grains
  • DDGS distiller's dried grain plus solubles
  • the fermentation end products can include without limitation glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids and derivatives thereof.
  • fermenting organisms are ethanologenic microorganisms or ethanol producing microorganisms such as ethanologenic bacteria which express alcohol dehydrogenase and pyruvate dehydrogenase and which can be obtained from Zymomonas moblis (See e.g. USP 5,000,000 ; USP 5,028,539 , USP 5,424,202 ; USP 5,514,583 and USP 5,554,520 ).
  • the ethanologenic microorganisms express xylose reductase and xylitol dehydrogenase, enzymes that convert xylose to xylulose.
  • xylose isomerase is used to convert xylose to xylulose.
  • a microorganism capable of fermenting both pentoses and hexoses to ethanol are utilized.
  • the microorganism can be a natural or non-genetically engineered microorganism or in other embodiments the microorganism can be a recombinant microorganism.
  • the fermenting microorganisms include, but not limited to, bacterial strains from Bacillus, Lactobacillus, E. coli, Erwinia, Pantoea (e.g., P. citrea), Pseudomonas and Klebsiella (e.g. K. oxytoca ). (See e.g. USP 5,028,539 , USP 5,424,202 and WO 95/13362 ). Bacillus is a preferred fermenting microorganism. The fermenting microorganism used in the fermenting step will depend on the end product to be produced.
  • the ethanol-producing microorganism can be a fungal microorganism, such as Trichoderma, a yeast and specifically Saccharomyces such as strains of S. cerevisiae ( USP 4,316,956 ).
  • S. cerevisiae A variety of S. cerevisiae are commercially available and these include but are not limited to FALI (Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM Specialties), (Ethanol Red, Fermentis, France), (SYNERXIA ® Thrive, Dupont), RED STAR (Lesaffre) and Angel alcohol yeast (Angel Yeast Company, China).
  • a Lactobacillus sp. L. casei
  • E. coli when glycerol or 1,3-propanediol are the desired end-products, E. coli can be used; and when 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired end products, Pantoea citrea can be used as the fermenting microorganism.
  • the above enumerated list are only examples and one skilled in the art will be aware of a number of fermenting microorganisms that can be appropriately used to obtain a desired end product.
  • the end product produced can be separated and/or purified from the fermentation media.
  • Methods for separation and purification are known, for example by subjecting the media to extraction, distillation and column chromatography.
  • the end product is identified directly by submitting the media to high-pressure liquid chromatography (HPLC) analysis.
  • HPLC high-pressure liquid chromatography
  • the mash can be separated by, for example, centrifugation into the liquid phase and solids phase and end products such as alcohol and solids recovered.
  • the alcohol can be recovered by means such as distillation and molecular sieve dehydration or ultrafiltration.
  • use of an enzyme blend or composition according to the invention in a method of ethanol production will result in a yield of ethanol that is greater than 8%, 10%, 12%, 14%, 16%, 17%, 18%, 19%, 20%, 21%, and 22% (v/v).
  • alcohol e.g. ethanol
  • Ethanol can be used for fuel, portable or industrial ethanol.
  • this method can be conducted wherein steps (b) and (c) are performed sequentially or simultaneously.
  • thermostable serine protease such as ME-3
  • the need for adding a nitrogen source during the saccharification or SSF process is reduced by at least at least 30%, 40%, 50, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or completely eliminated (100% reduction).
  • thermostable serine protease/metric ton (MT) starch-containing material wherein said thermostable serine protease has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3 is used.
  • thermostable serine protease/MT dry solid material wherein said thermostable serine protease has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs:3, 8, or 10 is used. It is believed that when step (b) and (c) are performed sequentially a similar reduction or elimination of a nitrogen source in step (b) can be observed.
  • a nitrogen source such as but not limiting to
  • thermostable serine protease/MT starch-containing material wherein said thermostable serine protease has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3 is used and, furthermore, when the fermentation product is ethanol then no acid proteolytic enzyme is needed in step (c).
  • thermostable serine protease having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3 in a method for producing fermentation products from starch-containing material can lead to additional benefits such as an increased level of crude protein, an increased level of acid hydrolyzed fat, a decreased level of glycerol (lower glycerol formation), a faster rate of ethanol production and an increased ethanol yield.
  • An increased level of hydrolyzed fat is indicative of increased recovery of oil.
  • Glycerol is a low value by-product of the fermentation that uses some of the available carbon. Reducing glycerol indicates more effective use of carbon and a reduced glycerol level indicates less stress on the yeast.
  • any reduction in the production of glycerol enables carbon to flow to higher value products such as ethanol and can lead to reduced viscosity.
  • a method for producing fermentation products from starch-containing material comprising:
  • a method for producing fermentation products from starch-containing material comprising:
  • a method for producing fermentation products from starch-containing material comprising:
  • a method for producing fermentation products from starch-containing material comprising:
  • Fermentation products contemplated hereunder can include alcohols (e.g., ethanol, methanol, butanol; polyols such as glycerol, sorbitol and inositol); organic acids, such as, citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid and the like; antibiotics, such as, pencillin and tetracycline; enzymes, vitamins, and hormones.
  • alcohols e.g., ethanol, methanol, butanol
  • polyols such as glycerol, sorbitol and inositol
  • organic acids such as, citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid and the like
  • antibiotics such as, pencillin and tetracycline
  • enzymes e.g., vitamins, and hormones.
  • the fermentation product can be ethanol, e.g., fuel ethanol; drinking ethanol such as potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry such as beer and wine, dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Beer types may comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high alcohol beer, low alcohol beer, low calorie beer or light beer. When ethanol is the fermentation product, it may be used as a fuel, which is typically blended with gasoline.
  • Starch to glucose processing generally consists of two steps and these steps include liquefaction of starch and saccharification of the liquefied starch. Further steps can include (a) purification and isomerization when the desired end product is a purified dextrose or fructose or (b) fermentation and distillation when the desired end product is, for example an alcohol ( e.g., ethanol).
  • steps include liquefaction of starch and saccharification of the liquefied starch. Further steps can include (a) purification and isomerization when the desired end product is a purified dextrose or fructose or (b) fermentation and distillation when the desired end product is, for example an alcohol (e.g., ethanol).
  • An object of the starch liquefaction process is to convert a slurry of starch polymer granules into a solution of shorter chain length dextrins of low viscosity. This is an important step for convenient handling of industrial equipment used in starch conversion processes.
  • the starch is liquefied by use of high temperature and enzymatic bioconversion.
  • a common enzymatic liquefaction process involves adding a thermostable bacterial alpha amylase (e.g .
  • SPEZYME ® PRIME and SPEZYME ® FRED SPEZYME ® ETHYL, SPEZYME ® RSL, SPEZYME ® CL, SPEZYME ® HT-WB from Danisco U.S., Inc, Genencor Division or TERMAMYL SC, TERMAMYL SUPRA or TERMANYL 120L from Novozymes) to a slurry comprising a substrate including starch and adjusting the pH to between 5.5 to 6.5 and the temperature to greater than 90°C.
  • SPEZYME ® RSL , SPEZYME ® CL and SPEZYME ® HT-WB can be added at a relevant commercial dose ranging from at least 0.05 to 5 kG/metric ton (MT) starch containing material, such as at least about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 kG/metric ton (MT) or any dose in between.
  • the starch is liquified and then subjected to saccharifying enzymes.
  • saccharification in the case of carbohydrate processing takes place in the presence of glucoamylase enzymes such as glucoamylase from Aspergillus niger (e.g., OPTIDEX L-400, DISTILLASE ® SSF, DISTILLASE ® CS, Spirizyme Fuel, Spirizyme Achieve) at a pH more acidic than the pH of the liquefaction step.
  • glucoamylase enzymes such as glucoamylase from Aspergillus niger (e.g., OPTIDEX L-400, DISTILLASE ® SSF, DISTILLASE ® CS, Spirizyme Fuel, Spirizyme Achieve) at a pH more acidic than the pH of the liquefaction step.
  • the fermentation product may be separated from the fermentation medium.
  • the mash (slurry) may be distilled to extract the desired fermentation product, e.g., ethanol.
  • the desired fermentation produce may be extracted from the medium by micro or membrane filtration techniques.
  • the fermentation product may also be recovered by stripping or other method well known in the art.
  • Lignocellulosic biomass may be treated by any method known by one skilled in the art to produce fermentable sugars in a hydrolysate.
  • the biomass may be pretreated using physical, thermal, or chemical treatments, or a combination thereof, and saccharified enzymatically.
  • Thermochemical pretreatment methods include steam explosion or methods of swelling the biomass to release sugars (see for example WO2010113129 ; WO2010113130 ).
  • Chemical saccharification may also be used.
  • Physical treatments may be used for particle size reduction prior to further chemical treatment. Chemical treatments include base treatment such as with strong base (ammonia or NaOH), or acid treatment ( US8545633 , WO2012103220 ).
  • the biomass can be pretreated with ammonia ( US 20080008783 , US 7932063 , US7781191 , US 7998713 , US7915017 ). These treatments release polymeric sugars from the biomass.
  • the pretreatment can be a low ammonia pretreatment where biomass is contacted with an aqueous solution comprising ammonia to form a biomass aqueous ammonia mixture where the ammonia concentration is sufficient to maintain alkaline pH of the biomass-aqueous ammonia mixture but is less than about 12 weight percent relative to dry weight of biomass.
  • the dry weight of biomass is at least about 15 weight percent solids relative to the weight of the biomass-aqueous ammonia mixture, as disclosed in the U.S. Patent No. 7,932,063 .
  • Saccharification which converts polymeric sugars to monomeric sugars, may be either by enzymatic or chemical treatments.
  • the pretreated biomass is contacted with a saccharification enzyme consortium under suitable conditions to produce a lignocellulosic biomass hydrolysate which comprises fermentable sugars.
  • the pretreated biomass Prior to saccharification, can be brought to the desired moisture content and treated to alter the pH, composition or temperature such that the enzymes of the saccharification enzyme consortium will be active.
  • the pH can be altered through the addition of acids in solid or liquid form.
  • carbon dioxide (CO2) which can be recovered from fermentation, can be utilized to lower the pH.
  • CO2 can be collected from a fermenter and fed into the pretreatment product headspace in the flash tank or bubbled through the pretreated biomass if adequate liquid is present while monitoring the pH, until the desired pH is achieved.
  • the temperature is brought to a temperature that is compatible with saccharification enzyme activity, as noted below.
  • suitable conditions can include temperature from about 40 °C to about 50 °C and pH between from about 4.8 to about 5.8.
  • Enzymatic saccharification of cellulosic or lignocellulosic biomass typically makes use of an enzyme composition or blend to break down cellulose and/or hemicellulose and to produce a hydrolysate containing sugars such as, for example, glucose, xylose, and arabinose. Saccharification enzymes are reviewed in Lynd, L. R., et al. (Microbiol. Mol. Biol. Rev., 66:506-577, 2002 ).
  • a saccharification enzyme blend can be used that includes one or more glycosidases.
  • Glycosidases hydrolyze the ether linkages of di-, oligo-, and polysaccharides and are found in the enzyme classification EC 3.2.1.x ( Enzyme Nomenclature 1992, Academic Press, San Diego, CA with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995, Supplement 4 (1997) and Supplement [in Eur. J. Biochem., 223:1-5, 1994 ; Eur. J. Biochem., 232:1-6, 1995 ; Eur. J. Biochem., 237:1-5, 1996 ; Eur. J. Biochem., 250:1-6, 1997 ; and Eur. J.
  • Glycosidases useful in saccharification can be categorized by the biomass components they hydrolyze. Glycosidases useful in saccharification can include cellulose-hydrolyzing glycosidases (for example, cellulases, endoglucanases, exoglucanases, cellobiohydrolases, b-glucosidases), hemicellulose-hydrolyzing glycosidases (for example, xylanases, endoxylanases, exoxylanases, b-xylosidases, arabino-xylanases, mannases, galactases, pectinases, glucuronidases), and starch-hydrolyzing glycosidases (for example, amylases, a-amylases, b-amylases, glucoamylase
  • a "cellulase" from a microorganism can comprise a group of enzymes, one or more or all of which can contribute to the cellulose-degrading activity.
  • Suitable enzymes may include enzymes from Glycoside hydrolase families, such as the families GH3, GH5, GH 6, GH 7, GH10, GH11, GH39, GH43, GH51, and GH 61.
  • GHs are a group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. Families of GHs have been classified based on sequence similarity and are available in the Carbohydrate-Active enzyme (CAZy) database ( Cantarel et al. (2009) Nucleic Acids Res. 37 (Database issue):D233-238 ).
  • Glycoside hydrolase family 3 (“GH3") enzymes have a number of known activities: b-glucosidase (EC:3.2.1.21); b-xylosidase (EC:3.2.1.37); N- acetyl b-glucosaminidase (EC:3.2.1.52); glucan b-1,3-glucosidase (EC:3.2.1.58); cellodextrinase (EC:3.2.1.74); exo-1,3-1,4-glucanase (EC:3.2.1); and b-galactosidase (EC 3.2.1.23).
  • Glycoside hydrolase family 39 (“GH39”) enzymes have a-L-iduronidase (EC:3.2.1.76) or b-xylosidase (EC:3.2.1.37) activity.
  • Glycoside hydrolase family 43 (“GH43”) enzymes have the following activities: L-25 a-arabinofuranosidase (EC 3.2.1.55); b-xylosidase (EC 3.2.1.37); endoarabinanase (EC 3.2.1.99); and galactan 1,3-b-galactosidase (EC 3.2.1.145).
  • Glycoside hydrolase family 51 (“GH51”) enzymes have L-a-arabinofuranosidase (EC 3.2.1.55) or endoglucanase (EC 3.2.1.4) activity.
  • Glycoside hydrolase family 10 (“GH10") are described, for example, in Schmidt et 30 al., 1999, Biochemistry 38:2403-2412 and Lo Leggio et al., 2001, FEBS Lett 509: 303-308 ) and the Glycoside hydrolase family 11 (“GH11") are more fully described in Hakouvainen et al., 1996, Biochemistry 35:9617-24 .
  • Suitable enzymes may include glycoside hydrolase family 61 ("GH61") enzymes which have been reclassified into Auxiliary Activity Family 9 (AA9; Merriypedia.org) and may be referred to as GH61 enzymes herein. As described in U.S.
  • suitable enzymes may include EG4 (such as from Trichoderma reesei and variants thereof as described, for example, in WO201517256A1 , WO201517255A1 , WO201517254A1 ), Fv3A, Fv51A and/or Fv43D (such as from Fusarium verticilloides as described, for example in US2014/016408 ).
  • Suitable enzymes may include, for example, enzymes disclosed in PCT Application Publication Nos.
  • WO03/027306 WO200352118_A2 , WO200352054_A2 , WO200352057_A2 , WO200352055_A2 , WO200352056_A2 , WO200416760_A2 , WO9210581_A1 , WO200448592_A2 , WO200443980_A2 , WO200528636_A2 , WO200501065_A2 , WO2005/001036 , WO2005/093050 , WO200593073_A1 , WO200674005_A2 , WO2009/149202 , WO2011/038019 , WO2010/141779 , WO2011/063308 , WO2012/125951 , WO2012/125925 , WO2012125937 , WO/2011/153276 , WO2014/093275 , WO2014/070837 , WO2014/0708
  • Saccharification enzymes can be obtained commercially. Such enzymes include, for example, SPEZYME ® CP cellulase, Multifect ® xylanase, Accelerase ® 1500, Accellerase ® DUET, Accellerase ® TrioTM, ® CS (from DuPont), DISTILLASE ® SSF, and Novozyme-188 (Novozymes). DISTILLASE ® SSF can be dosed at a commercial relevant dose of 0.06 to 0.08% weight enzyme / weight as is corn. The actual dose required will depend upon the conditions of the fermentation: time, initial pH and the level of solids.
  • DISTILLASE ® CS enzyme can be added at a commercial relevant dose of 0.055 to 0.075 % w/w (starch, dry solid basis). This corresponds to a level of 0.375 to 0.50 kg DISTILLASE ® CS / MT grain 'as is'. Additional enzymes can be added such as but not limiting to trehalase.
  • saccharification enzymes can be provided as crude preparations of a cell extract or a whole cell broth.
  • the enzymes can be produced using recombinant microorganisms that have been engineered to express one or more saccharifying enzymes.
  • an H3A protein preparation that may be used for saccharification of pretreated lignocellulosic biomass is a crude preparation of enzymes produced by a genetically engineered strain of Trichoderma reesei, which includes a combination of cellulases and hemicellulases and is described in WO 2011/038019 .
  • the enzyme may be introduced with or separate from the saccharification enzyme consortium.
  • Chemical saccharification treatments can be used and are known to one skilled in the art, such as treatment with mineral acids including HCl and H2SO4 ( US5580389 , WO2011002660 ).
  • Sugars such as glucose, xylose and arabinose are released by saccharification of lignocellulosic biomass and these monomeric sugars provide a carbohydrate source for a biocatalyst used in a fermentation process.
  • the sugars are present in a biomass hydrolysate that is used as fermentation medium.
  • the fermentation medium can be composed solely of hydrolysate, or can include components additional to the hydrolysate such as sorbitol or mannitol at a final concentration of about 5 mM as described in US 7,629,156 .
  • the biomass hydrolysate may make up at least about 50% of the fermentation medium. In embodiments, about 10% of the final volume of fermentation broth is seed inoculum containing the fermentation microorganism.
  • the fermentation medium comprising lignocellulosic biomass hydrolysate contacted with a fermentation microorganism is fermented in a fermentation vessel, which is any vessel that holds the fermentation medium and at least one biocatalyst, and has valves, vents, and/or ports used in managing the fermentation process.
  • a fermentation vessel which is any vessel that holds the fermentation medium and at least one biocatalyst, and has valves, vents, and/or ports used in managing the fermentation process.
  • Any microorganism that produces a target product utilizing glucose and preferably also xylose, either naturally or through genetic engineering, may be used for fermentation of the fermentable sugars in the lignocellulosic biomass hydrolysate.
  • Target products that may be produced by fermentation include, for example, acids, alcohols, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, and pharmaceuticals.
  • Alcohols include, but are not limited to methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propanediol, butanediol, glycerol, erythritol, xylitol, mannitol, and sorbitol.
  • Acids may include acetic acid, formic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, gluconic acid, itaconic acid, citric acid, succinic acid, 3- hydroxyproprionic acid, fumaric acid, maleic acid, and levulinic acid.
  • Amino acids may include glutamic acid, aspartic acid, methionine, lysine, glycine, arginine, threonine, phenylalanine and tyrosine.
  • Additional target products include methane, ethylene, acetone and industrial enzymes.
  • the fermentation of sugars in lignocellulosic biomass hydrolysate to target products can be carried out by one or more appropriate microorganisms, that are able to grow in medium containing biomass hydrolysate, in single or multistep fermentations.
  • Suitable microorganisms may be selected from bacteria, filamentous fungi and yeast.
  • the suitable microorganisms can be wild type microorganisms or recombinant microorganisms, and can include, for example, 15 organisms belonging to the genera of Escherichia, Zymomonas, Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus, and Clostridiuma.
  • Suitable microorganisms include recombinant Escherichia coli, Zymomonas mobilis, Bacillus stearothermophilus, Saccharomyces cerevisiae, Clostridia thermocellum, Thermoanaerobacterium saccharolyticum, and Pichia stipitis.
  • a microorganism can be selected or engineered to have higher tolerance to inhibitors present in biomass hydrolysate such as acetate.
  • the biocatalyst may produce ethanol as a target product, such as production of ethanol by Zymomonas mobilis, or production of ethanol by Saccharomyces cerevisiae.
  • Suitable engineered Zymomonas mobilis and Saccharomyces cerevisiae strains are known in the art, for example as described in US 8,247,208 and US 8,669,076 .
  • Fermentation is carried out with conditions appropriate for the particular microorganism used. Adjustments can be made for conditions such as pH, temperature, oxygen content, and mixing. Conditions for fermentation of yeast and bacterial biocatalysts are well known in the art.
  • saccharification and fermentation may occur at the same time in the same vessel, called simultaneous saccharification and fermentation (SSF).
  • SSF simultaneous saccharification and fermentation
  • partial saccharification may occur prior to a period of concurrent saccharification and fermentation in a process called HSF (hybrid saccharification and fermentation).
  • HSF hybrid saccharification and fermentation
  • a smaller culture (seed culture) of the fermentation microorganism is first grown. The seed culture is added to the fermentation medium as an inoculum typically in the range from about 2% to about 20% of the final volume.
  • fermentation by the fermentation microorganism produces a fermentation broth containing the target product made by the organism.
  • the fermentation broth may be a beer containing from about 6% to about 10% ethanol.
  • the fermentation broth contains water, solutes, solids from the hydrolysate medium and from biocatalyst metabolism of sugars in the hydrolysate medium, as well as the biocatalyst itself.
  • the target product may be isolated from the fermentation broth producing a depleted broth, which can be called whole stillage.
  • the broth is distilled, typically using a beer column, to generate an ethanol product stream and a whole stillage. Distillation can be using any conditions known to one skilled in the art including at atmospheric or reduced pressure.
  • the distilled ethanol is further passed through a rectification column and molecular sieve to recover an ethanol product.
  • the target product may alternatively be removed in a later step such as from a solid or liquid fraction after separation of fermentation broth.
  • thermostable serine protease it is possible to add an alpha-amylase during liquefaction along with the thermostable serine protease described herein.
  • a carbohydrate-source generating enzyme such as a glucoamylases, and/or optionally a pullulanase. Any alpha-amylase may be used.
  • Alpha-amylases and pullulanases are described above.
  • Biomass for second generation bioethanol production needs pretreatment before subjecting to enzyme hydrolysis to produce fermentable sugar. Biomass is usually pretreated at temperatures higher 100°C either under acidic or alkaline conditions ( DUNSON et al., TREATMENT OF BIOMASS TO OBTAIN FERMENTABLE SUGARS. WO2006110901 (A2) - 2006-10-19 ).
  • thermostable serine proteases like the thermostable serine proteases described herein may be used in such processes to hydrolyze any protein present in the biomass, to provide the ethanologen with amino acids and peptide nutrients, and to reduce the viscosity of the biomass slurry.
  • Biomass pretreated under alkaline conditions such as with diluted ammonium ( WO2006110901 ), is especially suitable for hydrolysis with said thermostable serine proteases due to their high pH.
  • thermostable serine protease having at least 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO:3.
  • oilseed fraction can be from soybean, corn seed, rape seed, palm kernel, sunflower seed, safflower seed, coconut, peanut, cotton seed, sesame seed, flax seed, poppy seed, almond, hazelnut, walnut, evening primrose seed, grape seed, hemp seed, black currant seed, red raspberry seed, carrot seed, cumin seed, blueberry seed, cranberry seed, parsley seed, onion seed, pumpkin seed, apricot kernel, mustard seed, linseed, castor seed or jatropha.
  • the oilseed fraction can comprise a protein fraction that is useful as a food, food ingredient, a food additive or food supplement.
  • plant-derived oil can be prepared by using the process described herein.
  • a feed, feedstuff, feed additive composition, premix, food or grain product can comprise the oil described herein.
  • Plant-derived lipids are a major source of lipids for food processing, and for industrial feedstock. More recently they are of interest and use as alternatives to petrochemicals for fuels.
  • Plant lipids can be derived from one or more parts of a plant, shrub, or tree. In various plants, the root, stem, bark, leaves, flowers, seeds, fruits, or other parts may serve as a source of oil.
  • Such lipids can be extracted mechanically, e.g. through the application of external pressure, or chemically, e.g. through organic or aqueous solvent extraction processes, or combination processes.
  • Oils obtained from oilseed plants are known for their uses in food, and food products, as well as for soaps, detergents, lotions, lubricants, insecticides, paints, coatings, inks, and other industrial or consumer products. Although the vast majority of oilseeds oils are extracted with an organic solvent extraction process, some are now extracted through aqueous extraction.
  • an “emulsion” comprises an at least transiently stable system comprising a physical mixture of at least two materials, not completely miscible with, or soluble in, each other.
  • Preferred emulsions, as used herein do not readily separate when they are allowed to stand undisturbed, and can remain mixed for considerable lengths of time. They preferably will remain stable for extended periods of time such as greater than about 1, 2, 4, 8 12, or 24 hours, or even longer.
  • the emulsions for use herein comprise at least a lipid phase and an aqueous phase, both of which are in the liquid state.
  • One phase in an emulsion is continuous, while the other phase is dispersed in and thus, discontinuous with, the other.
  • an "oil-in-water" emulsion has a discontinuous phase of oil dispersed in a continuous aqueous phase
  • a "water-in-oil” emulsion has a discontinuous aqueous phase dispersed in a continuous lipid or oil phase.
  • the discontinuous phase consists of small droplets dispersed in and contained within the other phase.
  • the discontinuous phase is thus also sometimes called the "internal” phase, and by analogy, the continuous phase is sometimes called the "external” phase.
  • an emulsion can invert, i.e. the continuous and discontinuous phases may change roles, for example, an oil-in-water emulsion becomes a water-in-oil emulsion or vice versa.
  • oil-in-water and “water-in-oil” as used herein are merely descriptive to help understand which of the phases is continuous and which is dispersed in a given emulsion system; these terms are not intended to limit the emulsion literally to oil and water.
  • the "water” or aqueous phase may contain one or more solutes, such as salts, and any number of other soluble, or partially soluble compounds.
  • the "oil” or lipidic phase may contain a wide variety of lipids or lipid-soluble compounds.
  • oil denotes any of a group of fats (or lipids) that remain liquid at room temperature.
  • Plant oil as used herein denotes any such oils that are obtained from any tissue of a plant. Plant oils are also referred to herein alternatively as “plant-derived oils”. In certain embodiments plant oils are edible, while in other embodiments they are not necessarily edible. Some oils may be appropriately and safely used for external application to an animal such as a human, while other oils may be completely inedible, and also not safe for external use on an animal. Such oils may nonetheless be valuable for use industrial process, as lubricants, cleaning or polishing products, or simply as feedstock for making other compositions or products requiring a lipid as a raw material. For example, some lipids are useful as fuels or fuel supplements. There is presently significant interest in biological or renewable sources of fuels, such as combustible fuels, for example lipids for use in biodiesel.
  • Oileed refers to any oil-containing seed, nut, kernel, or the like produced by a plant. All such plants, as well as their seeds, nuts, or kernels are contemplated for use herein.
  • the National Sustainable Agriculture Information Service lists the following as sources of oil for food, specialty, or industrial uses: almonds, apricot kernels, avocado, beech nut, billberry, black currant, borage, brazil nut, calendula, caraway seed, cashew nut, castor seed, citrus seed, clove, cocoa, coffee, copra (dried coconut), coriander, corn seed, cotton seed, elderberry, evening primrose, grape seed, groundnut, hazelnut, hemp seed, jojoba, linseed, macadamia nut, mace, melon seed, mustard seed, neem seed, niger seed, nutmeg, palm kernel, passion fruit, pecan, pistachio, poppy seed, pumpkin seed, rape seed, raspberry
  • oilseed and related plants whose oil content is of interest for use as fuel, such as "eco-fuel", biodiesel or the like.
  • Such plants include but are not limited to jatropha (e.g. Jatropha curcas, J. mahafalensis, and cultivars thereof); Elaeis guineensis (e.g. Oil palm), Aleurites fordii (tung oil tree or wood oil tree), Ricinus communis (castor bean tree), Copaifera langsdorfii (diesel tree), and Pongammia pinnata (Honge oil tree, or Pongam tree, and cultivars thereof).
  • jatropha e.g. Jatropha curcas, J. mahafalensis, and cultivars thereof
  • Elaeis guineensis e.g. Oil palm
  • Aleurites fordii tung oil tree or wood oil tree
  • Ricinus communis castor bean tree
  • Copaifera langsdorfii diese
  • oil-containing fraction or "lipid-containing fraction” as used herein refers to the oilseed or some portion or part thereof, however obtained.
  • the oil-containing fraction will comprise all, or at least a majority of, the oil (or fat or lipid content) of the oilseed.
  • a prior processing step may remove at least some, or even a majority of the oil from the oilseed prior to obtaining the fraction.
  • the "fraction” comprises soy flour or soy flakes.
  • the flour or flakes obtained are "full-fat" as that term is understood in the art.
  • the oil-containing fraction is from a major source of food or industrial oil.
  • soybean, corn seed, cotton seed, and rape seed, as well as sunflower seed, safflower, flax seed, and peanut are preferred as sources of food oil.
  • an "aqueous solvent” comprises at least water.
  • Aqueous solvents typically comprise other components such as salts, buffering compounds, small molecules, and more. Any number and concentration of additional components may be present provided the aqueous solvent is substantially a homogeneous solution or suspension in water.
  • This aqueous solvent, with the additional components as a substantially homogenous solution or suspension, is sometimes referred to herein as an "aqueous extractant" for convenience and to distinguish it from the solvent per se, e.g. water.
  • aqueous extractant is sometimes referred to herein as an "aqueous extractant" for convenience and to distinguish it from the solvent per se, e.g. water.
  • the two terms are synonymous as defined herein.
  • Preferably all components present are in true solution in the aqueous extractant or aqueous solvent.
  • aqueous solvent is distinguished from an organic solvent in that rather than water as the solvent, the term "organic solvent” refers to most other solvents that are organic compounds and contain carbon atoms. Organic solvents are more volatile and potentially explosive than aqueous solvents. Typically, organic solvent extraction of oils refers to any process used to remove an oil from oilseeds through direct contact with an organic solvent such as n-hexane or other hexanes.
  • thermostable serine protease having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs:3, 8, or 10, either alone or in combination with a phospholipase, under conditions allowing enzyme activity, for a time sufficient to destabilize the emulsion.
  • thermostable serine protease may be used in combinations with at least a phospholipase. While such enzyme activities from any source such as animal, plant, or microbial, are contemplated for use herein, the enzyme activity preferably comprises a phospholipase activity from a mammalian pancreas, Streptomyces violaceoruber, Aspergillus oryzae, or Aspergillus niger. In one embodiment, the phospholipase activity is from porcine pancreas.
  • the water phase is a continuous phase and the oil phase can be a discontinuous phase, i.e. the emulsion is an oil-in-water emulsion.
  • Processes for separating oil from aqueous phases are known in the art. Frequently, they involve gravity or more preferably forces in excess of gravity, for example forces applied through physical means such as centrifugation.
  • the emulsions are centrifuged in a batch-wise process.
  • the conditions for separating may include adjusting the emulsion after enzyme treatment to a preferred temperature, for example by heating.
  • continuous centrifugation is preferred for separating the oil from the aqueous phase. Continuous processes may be preferred to larger-scale operations and are well-suited to handling material by the vessel-full, for example, from silos, tanks, vats, or the like, or even in connected series of such vessels.
  • This process may improve the yield of oil from the emulsion.
  • the skilled artisan will appreciate how to monitor yield on a variety of bases.
  • the yield comparisons are made by comparing, for example, the yield of oil (for example on a % of theoretical maximum yield based on the oil content of the untreated emulsion) using the processes disclosed herein to the yield of an aqueous extraction that does not use the step of contacting the emulsion with the enzyme activity.
  • Other bases of yield may be used, for example, an improvement of oil recovery using the processes disclosed herein versus a "control" process.
  • the emulsion can be contacted with at least one enzyme activity comprising at least a thermostable serine protease activity as described herein either alone or in combination with a phospholipase. While such enzyme activities from any source such as animal, plant, or microbial, are contemplated for use herein, the enzyme activity preferably comprises a phospholipase activity from a mammalian pancreas, Streptomyces violaceoruber, Aspergillus oryzae, or Aspergillus niger. In one embodiment, the phospholipase activity is from porcine pancreas.
  • Phospholipases for purposes herein include, but are not limited to, phospholipases A (including A1 and A2), B (also sometimes referred to as lysophospholipase), C, and D.
  • Phospholipases are a class of enzymes that hydrolyze phospholipids, such as phosphatidylcholine or phosphatidylethanolamine. Within the phospholipase class of enzymes are five major subclasses, A1, A2, B, C, and D phospholipases.
  • A1 phospholipases (E.C. 3.1.1.32) preferentially hydrolyze the sn1 ester bonds of phospholipids, such as phosphatidylcholine or phosphatidylethanolamine, to yield 1-lysophospholipids plus carboxylic acids.
  • phospholipids such as phosphatidylcholine or phosphatidylethanolamine
  • A1 phospholipases require calcium as a cofactor.
  • A1 phospholipases generally exhibit broader specificity than A2 phospholipases.
  • A2 phospholipases (E.C. 3.1.1.4) preferentially hydrolyze the sn2 ester bonds of phospholipids, such as phosphatidylcholine or phosphatidylethanolamine, to yield 2-lysophospholipids plus carboxylic acids.
  • phospholipids such as phosphatidylcholine or phosphatidylethanolamine
  • A2 phospholipases show some specificity for hydrolysis of choline derivatives and phosphatides.
  • A2 phospholipases require calcium as a cofactor.
  • B phospholipases (E.C. 3.1.1.5) are also known as lysophospholipases. They preferentially hydrolyze the sn1 ester bonds of 2-lysophospholipids to yield glycerophosphatides plus carboxylic acids. B phospholipases will also hydrolyze the sn2 ester bonds of 1-lysophospholipids.
  • C phospholipases (E.C. 3.1.4.3) preferentially hydrolyze the phosphate bonds of phospholipids, such as phosphatidylcholine or phosphatidylethanolamine, to yield the corresponding diacylglycerols and choline phosphates. In addition to hydrolysis of phospholipids, C phospholipases will also act on lysophospholipids.
  • D phospholipases (E.C. 3.1.4.4) preferentially hydrolyze the phosphate bond of phospholipids such as phosphatidylcholine or phosphatidylethanolamine to yield the corresponding phosphatidic acids and choline. In addition to hydrolysis of phospholipids, D phospholipases will also act on lysophospholipids.
  • Phospholipases can be used individually or in combination or mixtures of one or more activities of the same or different E.C. classifications, and from the same or different sources. Crude or partially purified enzyme preparations containing one or more phospholipase activities are suitable for use in some embodiments herein. Commercial sources of phospholipases are also suitable for use herein. For example, Genencor- A Danisco Division (Rochester, NY) offers LysoMax ® and G-ZYME ® G999 phospholipases, from bacterial and fungal sources, respectively. Phospholipase C is available commercially, for example, from Sigma (St. Louis, MO).
  • C phospholipases (E.C. 3.1.4.3) preferentially hydrolyze the phosphate bonds of phospholipids, such as phosphatidylcholine or phosphatidylethanolamine, to yield the corresponding diacylglycerols and choline phosphates. In addition to hydrolysis of phospholipids, C phospholipases will also act on lysophospholipids.
  • D phospholipases (E.C. 3.1.4.4) preferentially hydrolyze the phosphate bond of phospholipids such as phosphatidylcholine or phosphatidylethanolamine to yield the corresponding phosphatidic acids and choline. In addition to hydrolysis of phospholipids, D phospholipases will also act on lysophospholipids.
  • the oil-in-water emulsion can be contacted with enzyme activity comprising at least a thermostable serine protease either alone or in combination with phospholipase.
  • Protein compositions prepared by the methods described herein are also provided, as are food products, consumer products, and industrial feedstock comprising a protein prepared by the methods or processes disclosed herein.
  • Thermobifida cellulosilytica DSM 44535 a bacterial strain obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), was selected as a potential source for enzymes useful in various industrial applications.
  • the entire genome of this strain was sequenced using ILLUMINA ® sequencing by synthesis technology. Genome sequencing and assembly of the sequence data was performed by BaseClear (Leiden, The Netherlands). Contigs were annotated by BioXpr (Namur, Belgium).
  • Tce01961n encodes a protease, designated ME-3 that showed homology to serine proteases of other bacteria.
  • the nucleotide sequence of Tce01961n, encoding the protease ME-3, is set forth as SEQ ID NO:1.
  • the amino acid sequence of the ME-3 preproenzyme encoded by Tce01961n is set forth as SEQ ID NO:2.
  • the ME-3 preproenzyme has a signal peptide with a predicted length of 27 residues (amino acids 1-27 in SEQ ID NO:2) as determined using SignalP ( Emanuelsson et al. (2007) Nature Protocols, 2: 953-971 ).
  • the presence of a signal peptide sequence indicates that this serine protease is a secreted enzyme.
  • the enzyme has a pro-sequence, with a predicted length of 162 residues ( amino acids 28-189 in SEQ ID NO:2).
  • the pro-sequence prediction was based on knowledge of the pro-mature junction in homologous proteases such as Cellulomonadin ( Shaw et al., (2007) Acta Crystallogr Sect F Struct Biol Cryst Commun 63: 266-269 ) and Thermobifida fusca Protease A ( Kelch BA & Agard DA (2007) J Mol Biol. 370:784-795 ).
  • homologous proteases such as Cellulomonadin ( Shaw et al., (2007) Acta Crystallogr Sect F Struct Biol Cryst Commun 63: 266-269 ) and Thermobifida fusca Protease A ( Kelch BA & Agard DA (2007) J Mol Biol. 370:784-795 ).
  • the amino acid sequence of the fully processed mature enzyme, ME-3 (186 amino acids), is depicted in SEQ ID NO:3.
  • the ME-3 protease was produced in Bacillus subtilis using an expression cassette consisting of the B. subtilis aprE promoter, a modified B. subtilis aprE signal peptide sequence, a codon-optimized sequence encoding the pro-mature sequence of ME-3 protease, and a BPN' terminator.
  • the sequence of the modified aprE signal peptide sequence fused to the codon-optimized DNA sequence encoding the pro-mature sequence of ME-3 protease is depicted in SEQ ID NO:4.
  • the ME-3 expression cassette was cloned into the pHYT replicating shuttle vector and transformed into a suitable B. subtilis strain.
  • the pHYT vector was derived from pHY300PLK (Takara Bio Inc.) by adding a terminator after the tetracycline resistance gene using the Bst EII and Eco RI sites (terminator sequence is set forth as SEQ ID NO:5).
  • the Hin dIII site in pHY300PLK was also removed using a linker cloned into the Bam HI and Hin dIII sites (linker sequence is set forth as SEQ ID NO:6).
  • a map of the pHYT vector for expression of the ME-3 protease (pHYT-Tce01961) is shown in Figure 1 .
  • a 96-well tray containing 150 ⁇ L tryptone soy broth (TBS) + 15 ppm tetracycline in each well was inoculated with a B. subtilis transformant containing pHYT-Tce01961.
  • the 96-well tray was incubated at overnight at 37°C, 200rpm in a shaking incubator. 30 ⁇ L of this overnight (pre)culture was added to a well in a 24-well tray containing 3 mL of enriched semi-defined media (based on MOPs buffer, with urea as major nitrogen source, glucose as the main carbon source, and supplemented with 1% soytone) supplemented with 5 mM CaCl 2 and 25 ppm tetracycline. The 24-well tray was incubated 32°C, 200 rpm for 48 hours.
  • the culture was harvested by centrifugation at 4500 rpm for 20 minutes.
  • the resulting supernatant was filtered through a 0.22 ⁇ m filter to produce a cell-free supernatant.
  • the supernatant contained ME-3 protease (confirmed by SDS-PAGE) and this fraction was further used for assays, and in some instances was further purified as described below.
  • ME-3 protease was purified by incubating the cell-free supernatant for 1 hour at 70°C in a water batch.
  • the incubated supernatant was centrifuged at 5100g for 10 minutes and subsequently filtered through a 0.22um filter. 0.02% azide was added and the sample was buffer exchanged using Viva spin 20ml ultrafiltration tubes of 10.000 MWCO (Sartorius). Centrifugation was performed to 5-10% of the original volume and 25mM HEPES buffer pH 7.0, 1mM CaCl 2 , 0.02% azide was added to a final volume of 20mL. This step was repeated 3 times and finally the sample was recovered from the ultrafiltration tube and 25mM HEPES buffer pH 7.0, 1mM CaCl 2 , 0.02% azide was added to obtain the desired final volume.
  • B. subtilis strain containing ME-3 expression cassette was cultured in 2 mL LB broth at 37°C for 8 hrs under antibiotic selection. A 100 ⁇ L aliquot of this pre-culture was added to a 125 mL baffled flask containing 10 mL of enriched semi-defined media (based on MOPs buffer, with urea as major nitrogen source, glucose as the main carbon source, and supplemented with 1% soytone) supplemented with antibiotics. The flasks were incubated for 2 days at 37 ° C or 42 ° C with constant rotational mixing at 250 rpm. The culture supernatant was recovered by centrifugation (20,000 g for 1 min) and used for quantification of ME-3 protease using the Suc-AAPF-pNA substrate as described in Example 3 below.
  • the concentration of ME-3 protease was calculated using the standard curve generated using purified enzyme.
  • the average yield of ME-3 protease (3 replicates) from cultures grown at 37°C and 42°C is reported in Table 1 below. As shown, an increase of approximately 50% in the yield of ME-3 protease was obtained when cells were grown at 42°C compared to 37°C. Table 1. Relative expression levels of ME-3 serine protease in Bacillus subtilis cultured at 37 or 42° C. 37°C 42°C ME-3 yield (ppm) 540 ⁇ 74 807 ⁇ 86
  • Cornsoy feed flour (composition shown on Table 2) was sifted to a particle size less than 212 ⁇ m and suspended in water to 10% (w/w) slurry and pH adjusted to pH 3. 138 ⁇ L of this slurry was added to each well in 96 MTP well-plate. A 20 ⁇ L sample of ME-3 protease prepared in 50mM acetate pH 3 was added, then 10 ⁇ Lporcine pepsin (Sigma, P7000, prepared at 800 U/mL in water) was added. The plate was incubated at 40°C for 45 min in iEMS microplate shaker incubator (iEMS) at 1150 rpm.
  • iEMS iEMS microplate shaker incubator
  • porcine pancreatin (Sigma P7545, prepared as 0.463 mg/mL in 1M Na-bicarbonate) was added and the plate was incubated at 40°C for 60 minutes in iEMS at 1150 rpm. Afterwards, the plate was centrifuged at 5°C, 4000 rpm for 15 min, 20 ⁇ L supernatant was transferred to new plates (coming plate #3641 non-binding) containing 180 ⁇ L water in each well to a 10x dilution.
  • the degree of hydrolysis (DH) of protein is based on the reaction of primary amino groups with ortho-phthaldialdehyde (OPA assay) as described by P.M. Nielsen, D. Petersen and C. Dambmann. Improved Method for Determining Food Protein Degree of Hydrolysis. Journal of Food Science. 66 (2001) 642-646 . This OPA assay was used to determine extent of hydrolysis.
  • the substrate Suc-AAPF-pNA (Sigma, S-7388) was used. The activity was measured as follows: 5 ⁇ L supernatant containing enzyme, 50 ⁇ L 0.2M Mes-NaOH (pH6.0), 50 ⁇ L water or 55 ⁇ L water (blank), 5 ⁇ L Suc-AAPF-pNA (20 mg/mL) were added to a half bottom area of 96 well microplate (Costar 3695, Corning Inc. NY, USA). The extent of hydrolysis was followed at 410 nm for up to 20 min.
  • feed samples were ground using a Perten laboratory mill with a particle size ⁇ 0.75 ⁇ m selection. 5 gram of the ground feed was dissolved in 50 mL of 0.1 M Tris pH 10 solution containing 1% (w/v) SDS, and centrifuged. The supernatant was filtered and used for detection of protease activity using the AAPF-pNA substrate and method described above.
  • the composition of cornsoy feed as presented in the Table 2 was derived from the following reference: Interactions of phytate and myo-inositol phosphate esters (IP 1-5 ) including IP 5 isomers with dietary protein and iron and inhibition of pepsin. S.
  • the thermal stability of ME-3 was also measured at pH 4.5 and pH 5.5 using 0.1M acetate It was found that after incubation at 85.5°C for 10min, the ME-3 enzyme retains about 85% activity at pH 4.5 and 5.5, while during incubation at 100°C for 5 min, the residual activity at pH 5.5 is about 65%.
  • the stability of the ME-3 protease during pelleting of animal feed was evaluated by measuring the percent enzyme activity recovery after the procedure. Pelleting of feed was performed at different temperatures at the Danish Technological Institute (Kolding, Denmark) according to standard operational procedures.
  • ME-3 enzyme without coating was diluted into 600 mL of water, and then sprayed directly onto 120 Kg of feed. This feed sample with sprayed-on ME-3 was pelleted at either 90, 95 or 100°C for 60 seconds.
  • the pellets were ground using a Perten laboratory mill and extracted in 50 mL of 0.1 M Tris pH 10 solution containing 1% (w/v) SDS at 22°C for 20min. The extract was centrifuged and filtered through filter paper.
  • the filtrate then used to determine the catalytic activity of ME-3 using the AAPF-pNA substrate assay described in Example 3. As shown on Figure 5 , the ME-3 enzyme without any coating retains more than 70% activity after pelleting at 90°C to 100°C for 60 seconds.
  • the ME-3 protease was added to a final concentration of 5ug/gDS and the 2kG mixture was incubated at 85°C for two hours under constantly stirring using an overhead mixer.
  • a control sample was generated by adding no protease to the liquefaction.
  • the treated corn sample (Liquefact) was collected and used in subsequent simultaneous saccharification and fermentation (SSF) experiments, and aliquots were also analyzed by HPLC size exclusion to determine the extent of corn liquefaction. Viscosity analysis during corn liquefaction.
  • Corn kernels (Arie Blok Animal Nutrition, NL-3440 AA Woerden, Artnr: 3777) were milled using a Retsch ZM200 grinding machine, settings: 3mm screen, 10k rpm.
  • the milled corn flour was used to generate a 25 gram 32% dry solids corn slurry by adding a 1:1 mixture of tap water/demineralized water to the flour.
  • the pH was adjusted to 5.5 with sulfuric acid and afterwards SPEZYME ® CL (DuPont commercial product containing a bacterial alpha amylase) was added at a commercial relevant dose.
  • SPEZYME ® CL DuPont commercial product containing a bacterial alpha amylase
  • the pH of the liquefact samples (with and without protease added to liquefaction reaction) were adjusted to 4.8 and DISTILLASE ® SSF (DuPont commercial product containing a fungal alpha amylase, a fungal glucoamylase, and an acid fungal protease) was added at a commercial relevant dose. Afterwards, 0.1% w/w active dry yeast (Ethanol Red, Fermentis, France) was added. A 50gram sample of this mixture was aliquoted into SSF vessels. Zero or 0.178 SAPU/g DS of FERMGEN TM 2.5x (DuPont commercial product containing an acid fungal protease) and various amounts of urea (0, 200 and 600 ppm) were added. The experiments were performed in duplicate. The fermentation incubations were performed using two different methods described below.
  • HPLC Analogenex Technologies 1200 series
  • run conditions were as follows: Phenomenex Rezex TM RFQ-Fast Acid H+ column held at 80°C, run at 1.0mL/min isocratic flow of 0.01N H 2 SO 4 solvent, a 10 ⁇ L injection volume, and 5.3 min elution runtime.
  • Refractive index detection was used for quantification of ethanol, and the results are shown in Table 4.
  • Table 4 shows that if urea dose is reduced from 600 to 200ppm (equivalent to 67% urea dose reduction) the rate of ethanol formation by liquefying enzymes such as SPEZYME RSL is slower.
  • ME-3 protease was added to the liquefaction mix including SPEZYME RSL, the fermentation rate is comparable to the one obtained when 600 ppm urea was included.
  • addition of ME-3 to liquefaction mix including SPEZYME RSL yielded that same ethanol yield when either zero or only 200 ppm urea were used instead. Therefore, ME-3 addition during liquefaction allows for a significant reduction, or elimination in urea requirement.
  • SSF Method 2 shows that if urea dose is reduced from 600 to 200ppm (equivalent to 67% urea dose reduction) the rate of ethanol formation by liquefying enzymes such as SPEZYME RSL is slower.
  • ME-3 protease was added to the liquefaction mix including SPEZYME RSL
  • the light grey line shows the ethanol yields using 240ppm urea and no FERMGEN TM 2.5x added to the fermentation and no ME-3 was used in the liquefaction.
  • the black line shows the ethanol yields when ME-3 protease was added to the liquefaction and the fermentation was performed with 240 ppm urea and no FERMGEN TM 2.5x addition.
  • ethanol yield was improved as compared to the control conditions.
  • HPLC-size exclusion was used to quantify corn protein solubilization.
  • the end-of-liquefaction samples were spun down at 13,000 rpm for 5 minutes and 200 uL of supernatant was filtrated through a 0.22um filter. A 5uL aliquot of this filtrate, was injected on an HPLC (Agilent Technologies 1200 series), on a Waters BEH125 ⁇ , 1.7um/300mm column; and run at 0.4mL/min.
  • Running buffer 25mM NaPO 4 pH6.8, 0.1M NaCl. Total area between retention time 7min to 10min was integrated, Detection: OD220nm.
  • results of the HPLC detection of peptides in liquefaction samples are shown in Table 5, as the integrated area under the curve with retention time between 7-10minutes for samples treated with and without ME-3 protease.
  • the protein hydrolysis results shown on Table 5 indicate that when ME-3 is added in liquefaction an increase in the levels of soluble peptides were observed.
  • Table 5 HPLC detection of peptides in liquefaction samples. Sample Relative area under the curve SPEZYME RSL 13,908 SPEZYME RSL + ME-3 35.990
  • Standard Brewing quality control analyses include the test for free amino nitrogen (FAN). This allows an estimation of the concentration of individual amino acids and small peptides which may be utilized by the yeast during beer fermentation for cell growth and proliferation.
  • FAN free amino nitrogen
  • the inclusion of non-malt raw materials as adjuncts in the brew process increase the requirements for higher FAN content in the wort to ensure proper fermentation process.
  • the protease ME-3 was tested in mashing operation with 50% Pilsner malt (Pilsner malt; Fuglsang Denmark, Batch 13.01.2016) and 50% corn grist (Benntag Nordic; Nordgetreide GmBH Lübec, Germany, Batch: 02.05.2016.), using a water to grist ratio of 4.0:1.
  • Pilsner malt was milled with a Buhler Miag malt mill (0.5 mm setting).
  • Maize grits (1.5g), Malt (milled pilsner malt, 1.5g) and tap water (12.0g) was mixed in Wheaton cups (Wheaton glass containers with cap) preincubated with 12.0g tap water pH adjusted to pH 5.4 with 2.5M sulphuric acid and heated to 70°C.
  • ME-3 protease was added based on ppm active protein (in total 0.5mL) and water as no enzyme control.
  • a commercially relevant dose of alpha-amylase (Amylex ® 5T, DuPont commercial product) was applied to all samples to facilitate liquefaction.
  • Wheaton cups were placed in a dry bath (Thermo Scientific Stem station) with magnetic stirring and the following mashing program was applied; kept at 70°C for 30 minutes; heated to 95°C for 12.5 minutes by increasing temperature with 2°C/minute; kept at 95°C for 17.5 minutes; cooled to 70°C for 15 minutes and held at 70°C for 30 minutes and finally heated to 79°C for 15 minutes and mashed off.
  • A10 mL sample was transferred to Falcon tubes and boiled at 100°C for 20 minutes to ensure complete enzyme inactivation.
  • Spend grains was separated from the wort by centrifugation in a Multifuge X3R centrifuge (Heraeus) at 4500 rpm for 30 minutes at 10°C.
  • Flaked, de-hulled and ground peanut seeds were suspended in 2mL 0.1M Tris-HCl containing 5mM CaCl 2 (pH8.0) and incubated at 60°C with shaking at 1180 ppm for 3 h to mimic the oil extraction process.
  • the oil water supernatant was collected by centrifugation at 4000 rpm for 15min using a benchtop centrifuge. The collected supernatant was treated with ME-3 protease to hydrolyze the proteins partitioned in the oil water emulsion.
  • thermostable protease like ME-3 is more compatible for the high temperature oil extraction process from plant and algae than those proteases disclosed in patent US2010227042 which are less thermostable.
  • Lipases including phospholipases may also be used in the process, but the advantage of using a thermostable protease is that it hydrolyzes plant proteins including those that may cause allergenicity and proteinaceous inhibitors that negatively affect digestion, thus producing a more desirable product.
  • the efficiency observed for the ME-3 protease to hydrolyze keratin wool material may be due to its tolerance to high temperature, since keratin proteins unfold more readily at higher temperature.
  • the current process for making feather meal for animal feed and pet food usually involves the treatment of feathers (high in keratin content) with high temperature to kill possible pathogens and to denature feather proteins.
  • ME-3 protease could either be used alone or in combination with the high temperature treatment of feathers (as described in patent number US2015197783 ).
  • Biomass for second generation bioethanol production or other purposes requires a pretreatment before being subjected to enzyme hydrolysis to produce fermentable sugars.
  • Biomass is usually pretreated at elevated temperatures, typically higher than 100°C, either under acidic or alkaline conditions (as described in patent application WO2006110901 , WO2014202716 ).
  • thermostable protease such as ME-3 may be used in such a process to hydrolyze the proteins present in the biomass in order to provide the fermenting organisms such as ethanologen with additional amino acids and peptide nutrients, and also to reduce the viscosity of the biomass slurry to increase its fluidity.
  • Alkaline pretreated biomass such as dilute ammonia pretreatment (as described in patent WO2006110901 ) would be especially suitable for ME-3 treatment to generate these nutrients due to the higher pH optimum of ME-3.
  • corn stover (63% solids) pretreated with diluted ammonia and suspended in deionized water at 10% (w/v) was used. The pH of this slurry was 8.25.
  • the slurry was distributed into 12 tubes each containing 4mL slurry.
  • the samples were mixed and reaction was performed at 60°C for 4h.
  • the samples were then centrifuged and the supernatant collected was filtered through 0.45 ⁇ m filter and diluted 10 times with water.
  • the samples were used to measure free amino groups from proteins and amino acids, by reacting 20uL of the diluted sample with OPA reagent as described in Example 3.
  • the extent of protein hydrolysis from biomass treatment by ME-3 are shown on Table 8. From Table 8, it can be seen that free amino groups as indicated by OPA value increased with the addition of ME-3. Table 8.
  • ME-3 Use of ME-3 to hydrolyze proteins in biomass.
  • ME-3 dose ⁇ L 0 1 5 10 15 25 OPA 340nm (average) 0.431 0.449 0.491 0.483 0.485 0.541 Stdev 0.017 0.016 0.016 0.019 0.035 0.059 CV 4.0 3.5 3.2 4.0 7.2 11.0 Percentage (%) 100 104 114 112 112 125
  • Tfpa A homolog present in Thermobifida fusca, Tfpa, was first described by Kelch,B.A. and Agard,D.A. (2007) J. Mol. Biol. 370: 784-795 .
  • the accession numbers of Tfpa at NCBI is 2PFE. Accession numbers WP_011290931, WP_016188200 and SCV75185 are identical proteins (mature chains).
  • the NCBI protein accession number of the ME-3 protease homolog in Thermobifida halotolerans (Thpa) is WP_068687914.
  • the amino acid sequence of Thermobifida fusca Tfpa preproenzyme is set forth as SEQ ID NO:7.
  • the Tfpa preproenzyme has a signal peptide with a predicted length of 31 residues (amino acids 1-31 in SEQ ID NO:7).
  • the pro-sequence of Tfpa has a length of 151 residues (amino acids 32-182 in SEQ ID NO:7)
  • the amino acid sequence of the fully processed mature enzyme, Tfpa (186 amino acids), is depicted in SEQ ID NO:8.
  • the amino acid sequence of Thermobifida halotolerans WP_068687914 peptidase S1, Thpa is set forth as SEQ ID NO:9. Based on alignments with ME-3 protease and Tfpa preproenzyme, the sequence of WP_068687914 seems to lack part of the N-terminal signal peptide.
  • the predicted, partial signal peptide corresponds to 15 residues(amino acids 1-15 in SEQ ID NO:9).
  • the predicted pro-sequence of Thermobifida halotolerans WP_068687914 has a length of 152 residues (amino acids 16-167 in SEQ ID NO:9).
  • the amino acid sequence of the predicted fully proc essed mature enzyme, Thermobifida halotolerans WP_068687914, Thpa (186 amino acids) is depicted in SEQ ID NO:10. Additional homologs of ME-3 protease were found by BLAST P search of NCBI database that is outside the genus Thermobifida include the following S1 proteases: Nocardiopsis potens protease (WP_017594871), Marinactinospora thermotolerans (WP_078763344.1), Amycolatopsis marina (WP_091675221.1), Actinoalloteichus hymeniacidonis (WP_069846166.1), Nocardiopsis trehalosi (WP_067965505.1), Saccharothrix sp. NRRL B-16314 (WP_033441214.1). The percent sequence identity across all the mature enzyme sequences was calculated and are shown on Table 9.
  • NRRL B-16314 WP_033441214 (amino acids 191-437) is shown in Figure 8 .
  • the sequences were aligned with default parameters using the MUSCLE program from Geneious software (Biomatters Ltd.) ( Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucl. Acids Res. (2004) 32 (5): 1792-1797 ).
  • the effects of the ME-3 protease was evaluated on dry matter digestibility (DMD), gas production, starch digestibility and pH in an in-vitro batch fermentation system using dent corn as substrate (4 mm ground).
  • the enzyme efficacy was evaluated at three doses (0.25, 0.5, and 0.75 mg/g of feed substrate) in three separate runs with four replicates per enzyme-dose in each run.
  • the effectiveness of the ME-3 was determined on the rumen fermentation pattern after incubation of buffered rumen contents with substrate and enzyme in 160 mL serum vials for 7 h 39°C.
  • the enzyme sample Prior to placement in the bottles, the enzyme sample was diluted in 0.1 M citrate-phosphate buffer (pH 6.0) and added to 0.5 g of the ground dent corn in F57 bags ( Krueger, N. A., and A. T. Adesogan. 2008. Anim. Feed Sci. Tech. 145: 84-94 ; Goering, H. K. and P. J. Van Soest. 1970. Agric. Handbook No. 379. ARS USDA, Washington, DC, pp. 20 ).
  • the F57 filter bags were sealed with an Uline Tabletop Poly Bag Sealer (Impulse ® type AIE-200) then immediately placed in the fermentation vessels (160 mL serum bottles).
  • TMR total mixed ration
  • the TMR ingredient composition fed to the fistulated dairy cows based on dry matter consisted of 38.2% corn silage, 27.3% ground shelled corn, 14.5% soybean meal with 44% crude protein, 9.1% Citrus pulp, 4.5% Feedlot premix, 4.0% alfalfa hay mid bloom, 1.8% energy booster (MS Specialty Nutrition, Dundee, IL), 0.5% Novasil (BASF, Germany), 100% in total.
  • the composition of artificial saliva included a micromineral solution of CaCl 2 ⁇ 2H 2 O, NMCl 2 ⁇ 4H 2 O, CoCl 2 ⁇ 6H 2 O, FeCl 2 ⁇ 6H 2 O; a macromineral solution of Na 2 HPO 4 ⁇ 12H 2 O, KH 2 PO 4 , MgSO 4 ⁇ 7H 2 O; a buffer solution of (NH 4 ) 2 HCO 3 and NaHCO 3 ; a trypticase peptone solution (tryptone, Sigma-Aldrich, St Louise, MO, USA); oxidation-reduction indicator resazurin and a reducing solution containing cysteine HCl, 1 M NaOH, Na 2 S ⁇ 9H 2 O and distilled water.
  • the volume ratio between the rumen fluid and the artificial saliva is 1:2.
  • thermostable serine protease ME-3 was further tested in lab scale corn liquefaction and simultaneous saccharification and fermentation (SSF).
  • Milled corn flour from a commercial ethanol plant was used to generate a slurry at 33% dry solids by adding a 6:4 w/w mixture of tap water:backset from a commercial ethanol plant to the flour.
  • the pH was adjusted to 5.1 with H 2 SO 4 and afterwards SPEZYME ® HT-WB (DuPont commercial product containing a bacterial alpha amylase) was dosed at a relevant commercial dose.
  • the mixture was incubated at 85°C for 20 minutes.
  • the pH of the liquefact samples (with and without ME-3 protease added to liquefaction reaction) was adjusted to 4.8 and a fungal alpha amylase, a fungal glucoamylase, and a fungal trehalase were added at a relevant dose to each SSF vessel containing 98 grams of liquefact.
  • 2mL of a propagated yeast culture (SYNERXIA ® Thrive, Dupont) was added and subsequently urea and FERMGEN TM 2.5x (DuPont commercial product containing an acid fungal protease) were added at different concentrations as indicated in Table 11.
  • the experiments were performed in quadruplicate.
  • the fermentation vessels were incubated at 32°C in a forced air incubator at 150rpm. Samples were collected at five different time points (16h, 24h, 40h, 48h and 54hours). These samples were analyzed for ethanol concentration, glycerol concentration, acid hydrolyzed fat and crude protein analysis as described below.
  • corn kernels (Arie Blok Animal Nutrition, NL-3440 AA Woerden, Artnr: 3777) were milled using a Retsch ZM200 grinding machine with settings: 3mm screen, 10k rpm.
  • the milled corn flour was used to generate a slurry at 32.64% dry solids by adding a 1:1 mixture of tap water/backset from a commercial ethanol plant to the flour.
  • the pH was adjusted to 5.5 with H 2 SO 4 and afterwards SPEZYME ® RSL (DuPont commercial product containing a bacterial alpha amylase) was dosed at a relevant commercial dose.
  • the ME-3 protease was added to a final concentration of 0, 3 or 6 ug/gDS and the mixture was incubated at 85°C for two hours under constantly stirring using an overhead mixer.
  • a control sample was generated by adding no protease to the liquefaction.
  • the treated corn sample (Liquefact) was collected and used in a subsequent SSF experiment.
  • the pH of the liquefact samples (with and without ME-3 protease added to liquefaction reaction) was adjusted to 4.8 and a fungal alpha amylase, a fungal glucoamylase, and a fungal trehalase were added at a relevant dose to each SSF vessel containing 98grams of liquefact.

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Claims (12)

  1. Verfahren zur Hydrolyse von Stärke enthaltendem Material, umfassend:
    (a) Inkontaktbringen von Stärke enthaltendem Material mit einer Flüssigkeit zur Bildung einer Maische; und
    (b) Hydrolyse von Stärke in der Maische zur Bildung eines Liquefacts mittels Inkontaktbringen der Maische mit einem Enzym-Cocktail, der eine thermostabile Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 umfasst.
  2. Verfahren nach Anspruch 1, wobei der Enzym-Cocktail ferner mindestens ein Enzym umfasst, das aus der Gruppe bestehend aus Alpha-Amylase, Amyloglucosidase, Phytase, Pullulanase, Beta-Glucanase, Cellulase und Xylanase ausgewählt ist.
  3. Verfahren zum Herstellen von Fermentationsprodukten aus Stärke enthaltendem Material, umfassend:
    (a) Verflüssigen des Stärke enthaltenden Materials mit einem Enzym-Cocktail, der eine thermostabile Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 umfasst;
    (b) Verzuckern des Produkts aus Schritt (a);
    (c) Fermentieren mit einem geeigneten Organismus; und
    (d) gegebenenfalls Entnehmen des in Schritt (c) hergestellten Produkts.
  4. Verfahren nach Anspruch 3, wobei Schritte (b) und (c) gleichzeitig durchgeführt werden.
  5. Verfahren nach Anspruch 3 oder Anspruch 4, wobei die Zugabe einer Stickstoffquelle durch Verwenden von 1-20 g thermostabiler Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 pro t Stärke enthaltendes Material eliminiert oder um mindestens 60 % reduziert wird.
  6. Verfahren nach Anspruch 5, wobei, wenn es sich bei dem Fermentationsprodukt um Ethanol handelt, in Schritt (c) bei der Verwendung von 1-20 g thermostabiler Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 pro t Stärke enthaltendes Material kein sauer proteolytisches Enzym benötigt wird.
  7. Verfahren zum Reduzieren von Viskosität eines verflüssigten Stärke enthaltenden Materials, das Inkontaktbringen einer Aufschlämmung des Stärke enthaltenden Materials mit einer thermostabilen Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 umfasst.
  8. Verfahren zum Extrahieren von Öl aus einer ölhaltigen Kulturpflanze, umfassend:
    (a) Inkontaktbringen einer Öl enthaltenden Ölsamenfraktion mit einem wässrigen Extraktionsmittel unter Bildung eines Extrakts der Ölsamenfraktion; und
    (b) Trennen der extrahierten Ölsamenfraktion in eine wässrige Phase, eine Öl-in-Wasser-Emulsion und eine unlösliche Phase;
    (c) Inkontaktbringen der Öl-in-Wasser-Emulsion mit mindestens einer thermostabilen Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 entweder alleine oder in Kombination mit mindestens einer Phospholipase unter Bedingungen, die Enzymaktivität über einen Zeitraum erlaubt, der zur Destabilisierung der Emulsion ausreicht; und
    (d) Trennen der destabilisierten Emulsion aus Schritt (c) in eine wässrige Phase, eine Öl-Phase und eine unlösliche Phase unter Erhalt von Öl aus dem Ölsamen.
  9. Verfahren nach Anspruch 8, wobei die Ölsamenfraktion von Sojabohne, Maissamen, Rapssamen, Palmkern, Sonnenblumensamen, Distelsamen, Kokosnuss, Erdnuss, Baumwollsamen, Sesamsamen, Flachssamen, Mohnsamen, Mandel, Haselnuss, Walnuss, Nachtkerzensamen, Traubenkern, Hanfsamen, Schwarze Johannisbeersamen, Himbeersamen, Karottensamen, Kümmelsamen, Blaubeersamen, Moosbeerensamen, Petersiliensamen, Zwiebelsamen, Kürbiskern, Aprikosenkern, Senfsamen, Leinsamen, Rizinussamen oder Jatropha stammt.
  10. Verfahren nach Anspruch 8, wobei die Ölsamenfraktion eine Proteinfraktion umfasst, die als Nahrungsmittel, Nahrungsmittelzutat, ein Nahrungszusatzmittel oder Nahrungsergänzungsmittel geeignet ist.
  11. Verfahren zur Hydrolyse von mindestens einem Nahrungsmittel- oder Tiernebenprodukt, umfassend:
    (a) Inkontaktbringen des Nahrungsmittel- oder Tiernebenprodukts mit einer Flüssigkeit; und
    (b) Hydrolyse des Nahrungsmittel- oder Tiernebenprodukts in der Maische zur Bildung eines Liquefacts durch Inkontaktbringen mit einem Enzym-Cocktail, der eine thermostabile Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 umfasst.
  12. Verfahren zur Hydrolyse von Proteinen in lignocellulosehaltiger Biomasse, umfassend
    (a) Inkontaktbringen der Biomasse mit einer Flüssigkeit; und
    (b) Hydrolyse der Proteine in der Biomasse mittels Inkontaktbringen der Biomasse mit einem Enzym-Cocktail, der eine thermostabile Serinprotease mit mindestens 95 % Sequenzidentität zu SEQ ID NO: 3 umfasst.
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BR112019012662A2 (pt) 2020-01-28
WO2018118815A1 (en) 2018-06-28
US20250136903A1 (en) 2025-05-01
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EP3558026A1 (de) 2019-10-30
CN110381746A (zh) 2019-10-25
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